Soluble ZcytoR14, anti-ZcytoR14 antibodies and binding partners and methods of using in inflammation

ABSTRACT

The present invention relates to blocking, inhibiting, reduceing, antagonizing or neutralizing the activity of IL-17F, IL-17A, or both IL-17A and IL-17F polypeptide molecules. IL-17A and IL-17F are cytokines that are involved in inflammatory processes and human disease. ZcytoR14 is a common receptor for IL-17A and IL-17F. The present invention includes soluble ZcytoR14, anti-ZcytoR14 antibodies and binding partners, as well as methods for antagonizing IL-17F, IL-17A or both IL-17A and IL-17F using such soluble receptors, antibodies and binding partners.

REFERENCE TO RELATED INVENTIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/578,805, filed Jun. 10, 2004, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains.

The demonstrated in vivo activities of cytokines and their receptorsillustrate the clinical potential of, and need for, other cytokines,cytokine receptors, cytokine agonists, and cytokine antagonists. Forexample, demonstrated in vivo activities of the pro-inflammatorycytokine family illustrates the enormous clinical potential of, and needfor antagonists of pro-inflammatory molecules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses these needs by providing antagonists topro-inflammatory cytokines IL-17A and IL-17F. Specifically, thepro-inflammatory cytokines IL-17A and IL-17F have a high degree ofsequence similarity, share many biological properties, and are bothproduced by activated T cells. They have both been implicated as factorsthat contribute to the progression of various autoimmune andinflammatory diseases including rheumatoid arthritis and asthma. Infact, reagents that negate IL-17A function significantly amelioratedisease incidence and severity in several mouse models of human disease.IL-17A mediates its effects through interaction with its cognatereceptor, the IL-17 receptor (IL-17R), but the receptor for IL-17F hasnot yet been identified. We now report that we have identified theIL-17R-related molecule, ZcytoR14 as the receptor for IL-17F. However,we have also noted that this receptor binds to both IL-17A and IL-17Fwith a similar high affinity. IL-17R on the other hand, binds IL-17Awith high affinity, but binds IL-17F with very low affinity. Consistentwith this, we have shown that a soluble form of IL-17R blocks IL-17Abinding and signaling in cells expressing either receptor, but does notinterfere with binding or function of IL-17F to ZcytoR14. In contrast, asoluble form of ZcytoR14 antagonizes both IL-17A and IL-17F, eithersingly or together, in cells expressing either receptor. Since IL-17Aintervention has been proposed as an effective therapy for severalauto-immune diseases, using antagonists of the present invention, whichmay block, inhibit, reduce, antagonize or neutralize the activity ofIL-17A, IL-17F, or both IL-17A and IL-17F, which include solubleZcytoR14 receptors and neutralizing anti-ZcytoR14 antibodies, will haveadvantages over therapies that target only one of these two cytokines.The invention further provides uses therefor in inflammatory disease, aswell as related compositions and methods.

A) Overview

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

A central event in both humoral and cell mediated immune responses isthe activation and clonal expansion of helper T cells. Helper T cellactivation is initiated by the interaction of the T cell receptor(TCR)—CD3 complex with an antigen-MHC on the surface of an antigenpresenting cell. This interaction mediates a cascade of biochemicalevents that induce the resting helper T cell to enter a cell cycle (theGO to G1 transition) and results in the expression of a high affinityreceptor for IL-2 and sometimes IL-4. The activated T cell progressesthrough the cycle proliferating and differentiating into memory cells oreffector cells.

In addition to the signals mediated through the TCR, activation of Tcells involves additional costimulation induced by cytokines released bythe antigen presenting cell or through interactions with membrane boundmolecules on the antigen presenting cell and the T cell. The cytokinesIL-1 and IL-6 have been shown to provide a costimulatory signal. Also,the interaction between the B7 molecule expressed on the surface of anantigen presenting cell and CD28 and CTLA4 molecules expressed on the Tcell surface effect T cell activation. Activated T cells express anincreased number of cellular adhesion molecules, such as ICAM-1,integrins, VLA4, LFA-1, CD56, etc.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In many immune responses, inflammatorycells infiltrate the site of injury or infection. The migrating cellsmay be neutrophilic, eosinophilic, monocytic or lymphocytic as can bedetermined by histologic examination of the affected tissues. CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,Inc.

Immune related diseases could be treated by suppressing the immuneresponse. Using soluble receptors and/or neutralizing antibodies thatinhibit molecules having immune stimulatory activity would be beneficialin the treatment of immune-mediated and inflammatory diseases. Moleculeswhich inhibit the immune response can be utilized (proteins directly orvia the use of antibody agonists) to inhibit the immune response andthus ameliorate immune related disease.

Interleukin-17 (IL-17A) has been identified as a cellular ortholog of aprotein encoded by the T lymphotropic Herpes virus Saimiri (HSV) [see,Rouvier et al., J. Immunol., 150(12): 5445-5456 (19993); Yao et al., J.Immunol., 122(12):5483-5486 (1995) and Yao et al., Immunity,3(6):811-821 (1995)]. Subsequent characterization has shown that thisprotein is a potent cytokine that acts to induce proinflammatoryresponses in a wide variety of peripheral tissues. IL-17A is adisulfide-linked homodimeric cytokine of about 32 kDa which issynthesized and secreted only by CD4+activated memory T cells (reviewedin Fossiez et al., Int. Rev. Immunol., 16: 541-551 [1998]).Specifically, IL-17 is synthesized as a precursor polypeptide of 155amino acids with an N-terminal signal sequence of 19-23 residues and issecreted as a disulfide-linked homodimeric glycoprotein. IL-17A isdisclosed in WO9518826 (1995), WO9715320 (1997) and WO9704097 (1997), aswell as U.S. Pat. No. 6,063,372.

Despite its restricted tissue distribution, IL-17A exhibits pleitropicbiological activities on various types of cells. IL-17A has been foundto stimulate the production of many cytokines. It induces the secretionof IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandinE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes,epithelial and endothelial cells. IL-17A also has the ability to induceICAM-1 surface expression, proliferation of T cells, and growth anddifferentiation of CD34.sup.+ human progenitors into neutrophils. IL-17Ahas also been implicated in bone metabolism, and has been suggested toplay an important role in pathological conditions characterized by thepresence of activated T cells and TNF-.alpha. production such asrheumatoid arthritis and loosening of bone implants (Van Bezooijen etal., J. Bone Miner. Res. 14: 1513-1521 [1999]). Activated T cells ofsynovial tissue derived from rheumatoid arthritis patients were found tosecrete higher amounts of IL-17A than those derived from normalindividuals or osteoarthritis patients (Chabaud et al., Arthritis Rheum.42: 963-970 [1999]). It was suggested that this proinflammatory cytokineactively contributes to synovial inflammation in rheumatoid arthritis.Apart from its proinflammatory role, IL-17A seems to contribute to thepathology of rheumatoid arthritis by yet another mechanism. For example,IL-17A has been shown to induce the expression of osteoclastdifferentiation factor (ODF) mRNA in osteoblasts (Kotake et al., J.Clin. Invest., 103: 1345-1352 [1999]). ODF stimulates differentiation ofprogenitor cells into osteoclasts, the cells involved in boneresorption.

Since the level of IL-17A is significantly increased in synovial fluidof rheumatoid arthritis patients, it appears that IL-17A inducedosteoclast formation plays a crucial role in bone resorption inrheumatoid arthritis. IL-17A is also believed to play a key role incertain other autoimmune disorders such as multiple sclerosis(Matusevicius et al., Mult. Scler., 5: 101-104 [19991). IL-17A hasfurther been shown, by intracellular signalling, to stimulate Ca.sup.2+influx and a reduction in [cAMP], in human macrophages (Jovanovic etal., J. Immunol., 160:3513 [1998]). Fibroblasts treated with IL-17Ainduce the activation of NF-.kappa.B, [Yao et al., Immunity, 3:811(1995), Jovanovic et al., supra], while macrophages treated with itactivate NF-.kappa.B and mitogen-activated protein kinases (Shalom-Bareket al., J. Biol. Chem., 273:27467 [1998]).

Additionally, IL-17A also shares sequence similarity with mammaliancytokine-like factor 7 that is involved in bone and cartilage growth.Other proteins with which IL-17A polypeptides share sequence similarityare human embryo-derived interleukin-related factor (EDIRF) andinterleukin-20.

Consistent with IL-17A's wide-range of effects, the cell surfacereceptor for IL-17A has been found to be widely expressed in manytissues and cell types (Yao et al., Cytokine, 9:794 [1997]). While theamino acid sequence of the human IL-17A receptor (IL-17R) (866 aminoacids) predicts a protein with a single transmembrane domain and a long,525 amino acid intracellular domain, the receptor sequence is unique andis not similar to that of any of the receptors from the cytokine/growthfactor receptor family. This coupled with the lack of similarity ofIL-17A itself to other known proteins indicates that IL-17A and itsreceptor may be part of a novel family of signalling proteins andreceptors. It has been demonstrated that IL-17A activity is mediatedthrough binding to its unique cell surface receptor, wherein previousstudies have shown that contacting T cells with a soluble form of theIL-17A receptor polypeptide inhibited T cell proliferation and IL-2production induced by PHA, concanavalin A and anti-TCR monoclonalantibody (Yao et al., J. Immunol., 155:5483-5486 [1995]). As such, thereis significant interest in identifying and characterizing novelpolypeptides having homology to the known cytokine receptors,specifically IL-17A receptors.

The expression pattern of IL-17F appears to be similar to that ofIL-17A, such that it includes only activated CD4+ T cells and monocytes(Starnes et al. J. Immunol. 167: 4137-4140 [2001]). IL-17F has beendemonstrated to induce G-CSF, IL-6, and IL-8 in fibroblasts (Hymowitz etal, EMBO J. 20:5322-5341 [2001]) and TGF-b in endothelial cells (Starneset al. J. Immunol. 167: 41374140 [2001]). It has recently been reportedthat IL-23, a cytokine produced by dendritic cell, can mediate theproduction of both IL-17A and IL-17F, primarily in memory T cells(Aggarwal et al. J. Biol. Chem. 278:1910-1914 [2003]).

Moreover, over expression or upregulation of both IL-17A and IL-17F havebeen shown in arthritic and asthmatic individuals (reviewed in Moseleyet al. CytokineGrowth Factor Rev 14:155-174 [2003]). With regards toarthritis, these cytokines act in a manner characteristic to thecartilage and joint destruction that is associated with rheumatoid- andosteo-arthritis. For example, IL-17A and IL-17F have been demonstratedto enhance matrix degradation in articular cartilage explants viarelease of cartilage proteoglycan glycosaminoglycans and collagenfragments, while inhibiting the synthesis of new proteoglycans andcollagens (Cai et al. Cytokine 16:10-21 [2001]; Attur et al ArthritisRheum 44:2078-2083 [2001]).

Similar to IL-17A, overexpression of IL-17F in mice has also been shownto increase lung neutrophil recruitment and result in increasedexpression of Th1-associated cytokines in the lung, including IL-6,IFN-gamma, IP-10 and MIG (Starnes et al. J. Immunol. 167: 41374140[2001]). IL-17F was also upregulated in T cells from allergen-challengedasthmatics (Kawaguchi et al J. Immunol 167:4430-4435 [2001]), and foundto induce IL-6 and IL-8 production in NHBE. In contrast to IL-17A,IL-17F appears to inhibit angiogenesis in vitro (Starnes et al. J.Immunol. 167: 4137-4140 [2001]).

IL-17F mRNA was not detected by northern blot in various human tissuesbut was dramatically induced upon activation of CD4+ T cells andmonocytes. Id. In mice, Th2 cells and mastr cells were found to expressIL-17F upon activation. See Dumont, Expert Opin. Ther. Patents 13(3)(2003). Like IL-17A, the expression of IL-17F was alos found to beupregulated by IL-23 in mouse.

The Il-17 cytokine/receptor families appear to represent a uniquesignaling system within the cytokine network that will offer innovativeapproaches to the manipulation of immune and inflammatory responses.Accordingly, the present invention is based on the discovery of a newIL-17 family receptor, ZcytoR14 and its ability to bind both IL-17A andIL-17F.

As such, antagonists to IL-17F and IL-17A activity, such as ZcytoR14soluble receptors and antibodies thereto including theanti-human-ZcytoR14 monoclonal and neutralizing antibodies of thepresent invention, are useful in therapeutic treatment of inflammatorydiseases, particularly as antagonists to both IL-17F and IL-17A singlyor together in the treatment of psoriasis. Moreover, antagonists toIL-17F activity, such as ZcytoR14 soluble receptors and antibodiesthereto including the anti-human-ZcytoR14 monoclonal and neutralizingantibodies of the present invention, are useful in therapeutic treatmentof other inflammatory diseases for example as bind, block, inhibit,reduce, antagonize or neutralize IL-17F and IL-17A (either individuallyor together) in the treatment of atopic and contact dermatitis, IBD,colitis, Endotoxemia, arthritis, rheumatoid arthritis, psoriaticarthritis, adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma, chronic obstructivepulmonary disease (COPD), airway hyper-responsiveness, chronicbronchitis, allergic asthma, bacterial pneumonia, psoriasis, eczema, andinflammatory bowel disease such as ulcerative colitis and Crohn'sdisease, helicobacter pylori infection. intraabdominal adhesions and/orabscesses as results of peritoneal inflammation (i.e. from infection,injury, etc.), systemic lupus erythematosus (SLE), multiple sclerosis,systemic sclerosis, nephrotic syndrome, organ allograft rejection, graftvs. host disease (GVHD), kidney, lung, heart, etc. transplant rejection,streptococcal cell wall (SCW)-induced arthritis, osteoarthritis,gingivitis/periodontitis, herpetic stromal keratitis, cancers includingprostate, renal, colon, ovarian, cervical, leukemia, angiogenesis,restenosis and kawasaki disease.

Cytokine receptors subunits are characterized by a multi-domainstructure comprising a ligand-binding domain and an effector domain thatis typically involved in signal transduction. Multimeric cytokinereceptors include monomers, homodimers (e.g., PDGF receptor αα and ββisoforms, erythropoietin receptor, MPL [thrombopoietin receptor], andG-CSF receptor), heterodimers whose subunits each have ligand-bindingand effector domains (e.g., PDGF receptor αβ isoform), and multimershaving component subunits with disparate functions (e.g., IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunitsare common to a plurality of receptors. For example, the AIC2B subunit,which cannot bind ligand on its own but includes an intracellular signaltransduction domain, is a component of IL-3 and GM-CSF receptors. Manycytokine receptors can be placed into one of four related families onthe basis of their structures and functions. Class I hematopoieticreceptors, for example, are characterized by the presence of a domaincontaining conserved cysteine residues and the WSXWS motif (SEQ IDNO:10). Additional domains, including protein kinase domains;fibronectin type III domains; and immunoglobulin domains, which arecharacterized by disulfide-bonded loops, are present in certainhematopoietic receptors. Cytokine receptor structure has been reviewedby Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. It is generally believed that under selective pressurefor organisms to acquire new biological functions, new receptor familymembers arose from duplication of existing receptor genes leading to theexistence of multi-gene families. Family members thus contain vestigesof the ancestral gene, and these characteristic features can beexploited in the isolation and identification of additional familymembers.

Amongst other inventions, the present invention provides novel uses fora soluble receptor, designated “ZcytoR14” or “soluble ZcytoR1438 or“sZcytoR14”, all of which may be used herein interchangeably, or andneutralizing antibodies to ZcytoR14 cytokine receptors. The presentinvention also provides soluble ZcytoR14 polypeptide fragments andfusion proteins, for use in human inflammatory and autoimmune diseases.The anti-ZcytoR14 antibodies, and soluble ZcytoR14 receptors of thepresent invention, including the neutralizing anti-ZcytoR14 antibodiesof the present invention, can be used to block, inhibit, reduce,antagonize or neutralize the activity of either IL-17F or IL-17A, orboth IL-17A and IL-17F in the treatment of inflammation and inflammatorydieases such as psoriasis, psoriatic arthritis, rheumatoid arthritis,endotoxemia, inflammatory bowel disease (IBD), colitis, asthma,allograft rejection, immune mediated renal diseases, hepatobiliarydiseases, multiple sclerosis, atherosclerosis, promotion of tumorgrowth, or degenerative joint disease and other inflammatory conditionsdisclosed herein.

An illustrative nucleotide sequence that encodes human ZcytoR14 isprovided by SEQ ID NO:1; the encoded polypeptide is shown in SEQ IDNO:2. ZcytoR14 functions as a receptor for both IL-17A (SEQ ID NOS:13 &14) and IL-17F (SEQ ID NOS:15 & 16). ZcytoR14 can act as a monomer, ahomodimer or a heterodimer. Preferably, ZcytoR14 acts as a homodimericreceptor for both IL-17A and/or IL-17F. ZcytoR14 can also act as aheterodimeric receptor subunit for a IL-17-related cytokine. ZcytoR14 isdisclosed in commonly owned U.S. patent application Ser. No. 10/458,647,and commonly owned WIPO publication WO 01/04304, both of which areincorporated herein in their entirety by reference. Analysis of a humancDNA clone encoding ZcytoR14 (SEQ ID NO:1) revealed an open readingframe encoding 692 amino acids (SEQ ID NO:2) comprising a putativesignal sequence of approximately 20 amino acid residues (amino acirdresidues 1 to 20 of SEQ ID NO:2), an extracellular ligand-binding domainof approximately 431 amino acid residues (amino acid residues 21-452 ofSEQ ID NO:2; SEQ ID NO:3), a transmembrane domain of approximately 20amino acid residues (amino acid residues 453-473 of SEQ ID NO:2), and anintracellular domain of approximately 203 amino acid residues (aminoacid residues 474 to 677 of SEQ ID NO:2). Furthermore, a ligand bindingdomain is represented by SEQ ID NO:22.

Yet another illustrative nucleotide sequence that encodes a varianthuman ZcytoR14, designated as “ZcytoR14-1” is provided by SEQ ID NO:4,the encoded polypeptide is shown in SEQ ID NO:5. ZcytoR14-1 is disclosedin commonly owned U.S. patent application Ser. No. 10/458,647, andcommonly owned WIPO publication WO 01/04304, both of which areincorporated herein in their entirety by reference. Sequence analysisrevealed that Zcytor14-1 is a truncated form of receptor polypeptide.That is, Zcytor14-1 lacks amino acid residues 1-113 of SEQ ID NO:2. SEQID NO:10 presents an amino acid sequence of a Zcytor14-1 polypeptidethat includes the N-terminal portion of Zcytor14.

A comparison of the Zcytor14 and Zcytor14-1 amino acid sequences alsoindicated that the two polypeptides represent alternatively splicedvariants. The amino acid sequence of Zcytor14 includes a 17 amino acidsegment (amino acid residues 339 to 355 of SEQ ID NO:2), whichZcytor14-1 lacks, while Zcytor14 lacks, following amino acid 479, a 13amino acid segment found in Zcytor14-1 (amino acid residues 350 to 362of SEQ ID NO:5). A polypeptide that contains both amino acid segments isprovided by SEQ ID NO:11, whereas SEQ ID NO:12 presents the amino acidsequence of a polypeptide that lacks both 13 and 17 amino acid segments.

Yet another illustrative nucleotide sequence that encodes a varianthuman ZcytoR14, designated as “ZcytoR14-6” is provided by SEQ ID NO:23,the encoded polypeptide is shown in SEQ ID NO:24. ZcytoR14-6 contains a25 amino acid residue deletion as compared to ZcytoR14 as embodied inSEQ ID NO:2. Specifically, ZcytoR14-6 does not contain amino acidresidue 94 to amino acid residue 118 of SEQ ID NO:2. Analysis of a humancDNA clone encoding ZcytoR14-6 (SEQ ID NO:23) revealed an extracellularligand-binding domain of approximately 427 amino acid residues (aminoacid residues 1-427 of SEQ ID NO:24), a transmembrane domain ofapproximately 20 amino acid residues (amino acid residues 428-448 of SEQID NO:24), and an intracellular domain of approximately 218 amino acidresidues (amino acid residues 449 to 667 of SEQ ID NO:24).

An illustrative nucleotide sequence that encodes a variant murineZcytoR14 is provided by SEQ ID NO:25; the encoded polypeptide is shownin SEQ ID NO:26. Murine ZcytoR14 functions as a receptor for both murineIL-17A (SEQ ID NOS:17 & 18) and murine IL-17F (SEQ ID NOS:19 & 20).Analysis of a murine cDNA clone encoding ZcytoR14 (SEQ ID NO:1) revealedan extracellular ligand-binding domain of approximately 449 amino acidresidues SEQ ID NO:27). Furthermore, a ligand binding domain isrepresented by SEQ ID NO:28.

Yet another illustrative nucleotide sequence that encodes a variantmurine ZcytoR14 is provided by SEQ ID NO:29; the encoded polypeptide isshown in SEQ ID NO:30.

The Zcytor14 gene resides in chromosome 3p25-3p24. As discussed below,this region is associated with various disorders and diseases.

Northern analyses indicate that there is strong expression of theZcytor14 gene in thyroid, adrenal gland, prostate, and liver tissues,and less expression in heart, small intestine, stomach, and tracheatissues. In contrast, there is little or no expression in brain,placenta, lung, skeletal muscle, kidney, pancreas, spleen, thymus,testis, ovary, colon, peripheral blood leukocytes, spinal cord, lymphnode, and bone marrow. These observations show that Zcytor14 sequencescan be used differentiate between various tissues.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 80%, orat least 90%, or greater than 95%, such as 96%, 97%, 98%, or greaterthan 99% or more identical to a reference amino acid sequence of 21-692of SEQ ID NO:2, wherein the isolated polypeptide specifically binds withan antibody that specifically binds with a polypeptide comprising theamino acid sequence of SEQ ID NO:2. The present invention also providesisolated polypeptides comprising an amino acid sequence that is at least70%, at least 80%, or at least 90% identical to a reference amino acidsequence selected from the group consisting of: (a) amino acid residues21 to 452 of SEQ ID NO:2, (b) amino acid residues 21 to 435 of SEQ IDNO:10, (c) amino acid residues 21 to 677 of SEQ ID NO:2, and (d) aminoacid residues 1 to 692 of SEQ ID NO:2, wherein the isolated polypeptidespecifically binds with an antibody that specifically binds with apolypeptide consisting of either the amino acid sequence of SEQ ID NO:2,or the amino acid sequence of SEQ ID NO:10. Illustrative polypeptidesinclude a polypeptide comprising the amino acid sequence of SEQ ID NO:2,SEQ ID NO:10, SEQ ID NO:1, or SEQ ID NO:12.

The present invention also provides isolated polypeptides comprising anextracellular domain, wherein the extracellular domain comprises eitheramino acid residues 21 to 452 of the amino acid sequence of SEQ ID NO:2or amino acid residues 21 to 435 of the amino acid sequence of SEQ IDNO:10. Such polypeptides may further comprise a transmembrane domainthat resides in a carboxyl-terminal position relative to theextracellular domain, wherein the transmembrane domain comprises aminoacid residues 453 to 473 of SEQ ID NO:2. These polypeptides may alsocomprise an intracellular domain that resides in a carboxyl-terminalposition relative to the transmembrane domain, wherein the intracellulardomain comprises either amino acid residues 474 to 677 of SEQ ID NO:2,or amino acid residues 457 to 673 of SEQ ID NO:10, and optionally, asignal secretory sequence that resides in an amino-terminal positionrelative to the extracellular domain, wherein the signal secretorysequence comprises amino acid residues 1 to 20 of the amino acidsequence of SEQ ID NO:2.

The present invention also includes variant Zcytor14 polypeptides,wherein the amino acid sequence of the variant polypeptide shares anidentity with the amino acid sequence of SEQ ID NO:2 selected from thegroup consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,and wherein any difference between the amino acid sequence of thevariant polypeptide and the amino acid sequence of SEQ ID NO:2 is due toone or more conservative amino acid substitutions.

Moreover, the present invention also provides isolated polypeptides asdisclosed above that bind IL-17F (e.g., human IL-17F polypeptidesequence as shown in SEQ ID NO:16). The human IL-17F polynucleotidesequence is shown in SEQ ID NO:15. The mouse IL-17F polynucleotidesequence is shown in SEQ ID NO:19, and corresponding polyepeptide isshown in SEQ ID NO:20. The present invention also provides isolatedpolypeptides as disclosed above that bind IL-17A (e.g., human IL-17Apolypeptide sequence as shown in SEQ ID NO:14). The human IL-17Apolynucleotide sequence is shown in SEQ ID NO:13. The mouse IL-17Apolynucleotide sequence is shown in SEQ ID NO:17, and correspondingpolyepeptide is shown in SEQ ID NO:18.

The present invention also provides isolated polypeptides and epitopescomprising at least 15 contiguous amino acid residues of an amino acidsequence of SEQ ID NO:2 or 3. Illustrative polypeptides includepolypeptides that either comprise, or consist of SEQ ID NO:2 or 3, anantigenic epitope thereof, or a functional IL-17A or IL-17F bindingfragment thereof. Moreover, the present invention also provides isolatedpolypeptides as disclosed above that bind to, block, inhibit, reduce,antagonize or neutralize the activity of IL-17F or IL-17A.

The present invention also includes variant ZcytoR14 polypeptides,wherein the amino acid sequence of the variant polypeptide shares anidentity with the amino acid residues of SEQ ID NO:2 selected from thegroup consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,such as 96%, 97%, 98%, or greater than 99% or more identity, and whereinany difference between the amino acid sequence of the variantpolypeptide and the corresponding amino acid sequence of SEQ ID NO:2 isdue to one or more conservative arnino acid substitutions. Suchconservative amino acid substitutions are described herein. Moreover,the present invention also provides isolated polypeptides as disclosedabove that bind to, block, inhibit, reduce, antagonize or neutralize theactivity of IL-17F or IL-17A.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude neutralizing antibodies, polyclonal antibodies, murinemonoclonal antibodies, humanized antibodies derived from murinemonoclonal antibodies, and human monoclonal antibodies. Illustrativeantibody fragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, andminimal recognition units. Neutralizing antibodies preferably bindZcytoR14 such that the interaction of IL-17A and IL-17F with ZcytoR14 isblocked, inhibited, reduced, antagonized or neutralized; anti-ZcytoR14neutralizing antibodies such that the binding of either IL-17A or IL-17Fto ZcytoR14 is blocked, inhibited, reduced, antagonized or neutralizedare also encompassed by the present invention. That is, the neutralizinganti-ZcytoR14 antibodies of the present invention can either eitherbind, block, inhibit, reduce, antagonize or neutralize each of IL-17A orIL-17F singly, or bind, block, inhibit, reduce, antagonize or neutralizeIL-17A and IL-17F together. The present invention further includescompositions comprising a carrier and a peptide, polypeptide, orantibody described herein.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide or antibody described herein.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or a fragment thereof. An exemplaryanti-idiotype antibody binds with an antibody that specifically binds apolypeptide consisting of SEQ ID NO:2.

The present invention also provides fusion proteins, comprising aZcytoR14 polypeptide and an immunoglobulin moiety. In such fusionproteins, the immunoglobulin moiety may be an immunoglobulin heavy chainconstant region, such as a human F_(C) fragment. The present inventionfurther includes isolated nucleic acid molecules that encode such fusionproteins.

The present invention also provides polyclonal and monoclonal antibodiesthat bind to polypeptides comprising an ZcytoR14 extracellular domainsuch as monomeric, homodimeric, heterodimeric and multimeric receptors,including soluble receptors. Moreover, such antibodies can be usedantagonize the binding of ZcytoR14 ligands, IL-17F (SEQ ID NO:16), andIL-17A (SEQ ID NO:14), individually or together to the ZcytoR 14receptor.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

B) Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called ¢peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

¢Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that producesZcytoR14 from an expression vector. In contrast, ZcytoR14 can beproduced by a cell that is a “natural source” of ZcytoR14, and thatlacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a ZcytoR14polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities ofZcytoR14 using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains,and other linkage to the cell membrane such as via glycophosphoinositol(gpi). Soluble receptors can comprise additional amino acid residues,such as affinity tags that provide for purification of the polypeptideor provide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Solublereceptors can be monomeric, homodimeric, heterodimeric, or multimeric,with multimeric receptors generally not comprising more than 9 subunits,preferably not comprising more than 6 subunits, and most preferably notcomprising more than 3 subunits. Receptor polypeptides are said to besubstantially free of transmembrane and intracellular polypeptidesegments when they lack sufficient portions of these segments to providemembrane anchoring or signal transduction, respectively. Solublereceptors of cytokine receptors generally comprise the extracellularcytokine binding domain free of a transmsmbrane domain and intracellulardomain. For example, representative soluble receptors include solublereceptors for IL-17R as shown in SEQ ID NO:21. It is well within thelevel of one of skill in the art to delineate what sequences of a knowncytokine receptor sequence comprise the extracellular cytokine bindingdomain free of a transmsmbrane domain and intracellular domain.Moreover, one of skill in the art using the genetic code can readilydetermine polynucleotides that encode such soluble receptor polyptides.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, an dthe like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of ananti-ZcytoR14 antibody, and thus, an anti-idiotype antibody mimics anepitope of ZcytoR14.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-ZcytoR14 monoclonal antibody fragmentbinds with an epitope of ZcytoR14.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain. Construction of humanizedantibodies for therapeutic use in humans that are derived from murineantibodies, such as those that bind to or neutralize a human protein, iswithin the skill of one in the art.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a ZcytoR14polypeptide component. Examples of an antibody fusion protein include aprotein that comprises a ZcytoR14 extracellular domain, and either an Fcdomain or an antigen-binding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major -histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for ZcytoR14” or a “ZcytoR14antisense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the ZcytoR14 gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the ZcytoR14 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant ZcytoR14 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of ZcytoR14 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of ZcytoR14 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant ZcytoR14 gene can be identified,for example, by determining whether the gene hybridizes with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1 OR SEQ IDNO:4, or its complement, under stringent conditions.

Alternatively, variant ZcytoR14 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant ZcytoR14gene or variant ZcytoR14 polypeptide, a variant gene or polypeptideencoded by a variant gene may be functionally characterized the abilityto bind specifically to an anti-ZcytoR14 antibody. A variant ZcytoR14gene or variant ZcytoR14 polypeptide may also be functionallycharacterized the ability to bind to its ligand, for example, IL-17Aand/or IL-17F, using a biological or biochemical assay described herein.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of ZcytoR14 genes.Within the context of this invention, a “functional fragment” of aZcytoR14 gene refers to a nucleic acid molecule that encodes a portionof a ZcytoR14 polypeptide which is a domain described herein or at leastspecifically binds with an anti-ZcytoR14 antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

C) Production of ZcytoR14 Polynucleotides or Genes

Nucleic acid molecules encoding a human ZcytoR14 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1 OR SEQ ID NO:4. These techniques are standard andwell-established, and may be accomplished using cloning kits availableby commercial suppliers. See, for example, Ausubel et al. (eds.), ShortProtocols in Molecular Biology, 3^(rd) Edition, John Wiley & Sons 1995;Wu et al., Methods in Gene Biotechnology, CRC Press, Inc. 1997; Aviv andLeder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Huynh et al.,“Constructing and Screening cDNA Libraries in λgt10 and λgt11,” in DNACloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press,1985); Wu (1997) at pages 47-52.

Nucleic acid molecules that encode a human ZcytoR14 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the ZcytoR14 gene or cDNA. General methods for screeninglibraries with PCR are provided by, for example, Yu et al., “Use of thePolymerase Chain Reaction to Screen Phage Libraries,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, White (ed.), Humana Press, Inc., 1993. Moreover,techniques for using PCR to isolate related genes are described by, forexample, Preston, “Use of Degenerate Oligonucleotide Primers and thePolymerase Chain Reaction to Clone Gene Family Members,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, White (ed.), Humana Press, Inc. 1993. As an alternative, aZcytoR14 gene can be obtained by synthesizing nucleic acid moleculesusing mutually priming long oligonucleotides and the nucleotidesequences described herein (see, for example, Ausubel (1995)).Established techniques using the polymerase chain reaction provide theability to synthesize DNA molecules at least two kilobases in length(Adang et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCRMethods and Applications 2:266 (1993), Dillon et al., “Use of thePolymerase Chain Reaction for the Rapid Construction of SyntheticGenes,” in Methods in Molecular Biology, Vol. 15: PCR Protocols: CurrentMethods and Applications, White (ed.), pages 263-268, (Humana Press,Inc. 1993), and Holowachuk et al., PCR Methods Appl. 4:299 (1995)). Forreviews on polynucleotide synthesis, see, for example, Glick andPasternak, Molecular Biotechnology, Principles and Applications ofRecombinant DNA (ASM Press 1994), Itakura et al., Annu. Rev. Biochem.53:323 (1984), and Climie et al., Proc. Nat'l Acad. Sci. USA 87:633(1990).

D) Production of ZcytoR14 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the ZcytoR14 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. Moreover,the present invention also provides isolated soluble monomeric,homodimeric, heterodimeric and multimeric receptor polypeptides thatcomprise at least one ZcytoR14 receptor subunit that is substantiallyhomologous to the receptor polypeptide of SEQ ID NO:2. Thus, the presentinvention contemplates ZcytoR14 polypeptide-encoding nucleic acidmolecules comprising degenerate nucleotides of SEQ ID NO:1 or SEQ IDNO:4, and their RNA equivalents.

Those skilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:7 is adegenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zcytor14 polypeptide of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:7 also provides all RNA sequences encoding SEQ ID NO:2, bysubstituting U for T. Thus, the present invention contemplates Zcytor14polypeptide-encoding nucleic acid molecules comprising nucleotide 154 tonucleotide 2229 of SEQ ID NO:1, and their RNA equivalents. Similarly,the Zcytor14-1 degenerate sequence of SEQ ID NO:6 also provides all RNAsequences encoding SEQ ID NO:5, by substituting U for T.

Table 1 sets forth the one-letter codes to denote degenerate nucleotidepositions. “Resolutions” are the nucleotides denoted by a code letter.“Complement” indicates the code for the complementary nucleotide(s). Forexample, the code Y denotes either C or T, and its complement R denotesA or G, A being complementary to T, and G being complementary to C.TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons, encompassing all possible codons for a givenamino acid, are set forth in Table 2. TABLE 2 One Amino LetterDegenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT TCATCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN AlaA GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp DGAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg RAGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATAATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTNPhe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter TAA TAG TGA TRRAsn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NO:3. Variant sequences can be readily tested forfunctionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed herein serve as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

A ZcytoR14-encoding cDNA can be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction with primers designedfrom the representative human ZcytoR14 sequences disclosed herein. Inaddition, a cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to ZcytoR14 polypeptide.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human ZcytoR14, and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequences disclosedherein, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of theamino acid sequences disclosed herein. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the ZcytoR14polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that comprise a soluble ZcytoR14receptor subunit that is substantially homologous to either SEQ ID NO:1or SEQ ID NO:4, or that encodes amino acids of either SEQ ID NO:2 or SEQID NO:5, or allelic variants thereof and retain the ligand-bindingproperties of the wild-type ZcytoR14 receptor. Such polypeptides mayalso include additional polypeptide segments as generally disclosedherein.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:1OR SEQ ID NO:4, or to nucleic acid molecules comprising a nucleotidesequence complementary to SEQ ID NO:1 OR SEQ ID NO:4, or fragmentsthereof.

In general, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Following hybridization, thenucleic acid molecules can be washed to remove non-hybridized nucleicacid molecules under stringent conditions, or under highly stringentconditions. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user-defined criteria. It is well within theabilities of one skilled in the art to adapthybridization and washconditions for use with a particular polynucleotide hybrid.

The present invention also provides isolated ZcytoR14 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95%, such as 96%, 97%, 98%, orgreater than 95% sequence identity to the sequences shown in SEQ IDNO:2, or their orthologs. For example, variant and orthologous ZcytoR14receptors can be used to generate an immune response and raisecross-reactive antibodies to human ZcytoR14. Such antibodies can behumanized, and modified as described herein, and used therauputically totreat psoriasis, psoriatic arthritis, IBD, colitis, endotoxemia as wellas in other therapeutic applications described herein.

The present invention also contemplates ZcytoR14 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay. Such ZcytoR14variants include nucleic acid molecules (1) that remain hybridized witha nucleic acid molecule having the nucleotide sequence of SEQ ID NO: IOR SEQ ID NO:4 (or its complement) under stringent washing conditions,in which the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDSat 55-65° C., and (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95%, or greater than 95% such as 96%,97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ IDNO:2. Alternatively, ZcytoR14 variants can be characterized as nucleicacid molecules (1) that remain hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 OR SEQ ID NO:4 (or itscomplement) under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and(2) that encode a polypeptide having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95%, such as 96%, 97%, 98%, or99% or greater, sequence identity to the amino acid sequence of SEQ IDNO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul el al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100). TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R−1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 25 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3−4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −25 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0−3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0−1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1−2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1−1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZcytoR14 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2 or SEQ ID NO:3) and a test sequence that have either thehighest density of identities (if the ktup variable is 1) or pairs ofidentities (if ktup=2), without considering conservative amino acidsubstitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using an amino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Illustrative parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNO:2, in which an alkyl amino acid is substituted for an alkyl aminoacid in a ZcytoR14 amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a ZcytoR14 amino acidsequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a ZcytoR14 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a ZcytoR14 amino acid sequence, an acidic amino acid issubstituted for an acidic amino acid in a ZcytoR14 amino acid sequence,a basic amino acid is substituted for a basic amino acid in a ZcytoR14amino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in a ZcytoR14 aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).Particular variants of ZcytoR14 are characterizedby having at least 70%, at least 80%, at least 90%, at least 95% orgreater than 95% such as 96%, 97%, 98%, or 99% or greater sequenceidentity to the corresponding amino acid sequence (e.g., SEQ ID NO:2),wherein the variation in amino acid sequence is due to one or moreconservative amino acid substitutions.

Conservative amino acid changes in a ZcytoR14 gene can be introduced,for example, by substituting nucleotides for the nucleotides recited inSEQ ID NO:1 OR SEQ ID NO:4. Such “conservative amino acid” variants canbe obtained by oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (see Ausubel (1995); and McPherson (ed.), Directed Mutagenesis: APractical Approach (IRL Press 1991)). A variant ZcytoR14 polypeptide canbe identified by the ability to specifically bind anti-ZcytoR14antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for ZcytoR14 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

Although sequence analysis can be used to further define the ZcytoR14ligand binding region, amino acids that play a role in ZcytoR14 bindingactivity (such as binding of ZcytoR14 to either Il-17A or IL-17F, or toan anti-ZcytoR14 antibody) can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., Science 255:306 (1992), Smith etal., J. Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett.309:59 (1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover, ZcytoR14labeled with biotin or FITC can be used for expression cloning ofZcytoR14 ligands.

Variants of the disclosed ZcytoR14 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNA molecules are generated by in vitro homologous recombination byrandom fragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-ZcytoR14 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of ZcytoR14polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a ZcytoR14 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 OR SEQ ID NO:4 can be digestedwith Bal31 nuclease to obtain a series of nested deletions. Thefragments are then inserted into expression vectors in proper readingframe, and the expressed polypeptides are isolated and tested for theability to bind anti-ZcytoR14 antibodies. One alternative to exonucleasedigestion is to use oligonucleotide-directed mutagenesis to introducedeletions or stop codons to specify production of a desired fragment.Alternatively, particular fragments of a ZcytoR14 gene can besynthesized using the polymerase chain reaction.

This general approach is exemplified by studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50.1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

The present invention also contemplates functional fragments of aZcytoR14 gene that have amino acid changes, compared with an amino acidsequence disclosed herein. A variant ZcytoR14 gene can be identified onthe basis of structure by determining the level of identity withdisclosed nucleotide and amino acid sequences, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant ZcytoR14 gene can hybridize to a nucleic acid moleculecomprising a nucleotide sequence, such as SEQ ID NO:1 OR SEQ ID NO:4.

The present invention also includes using functional fragments ofZcytoR14 polypeptides, antigenic epitopes, epitope-bearing portions ofZcytoR14 polypeptides, and nucleic acid molecules that encode suchfunctional fragments, antigenic epitopes, epitope-bearing portions ofZcytoR14 polypeptides. Such fragments are used to generate polypeptidesfor use in generating antibodies and binding partners that bind, block,inhibit, reduce, antagonize or neutralize activity of IL-17A or IL-17For both IL-17A and IL-17F. A “functional” ZcytoR14 polypeptide orfragment thereof as defined herein is characterized by its ability toblock, inhibit, reduce, antagonize or neutralize IL-17A or IL-17Finflammatory, proliferative or differentiating activity, by its abilityto induce or inhibit specialized cell functions, or by its ability tobind specifically to an anti-ZcytoR14 antibody, cell, IL-17A or IL-17F.As previously described herein, ZcytoR14 is characterized by a uniquecytokine receptor structure and domains as described herein. Thus, thepresent invention further contemplates using fusion proteinsencompassing: (a) polypeptide molecules comprising one or more of thedomains described above; and (b) functional fragments comprising one ormore of these domains. The other polypeptide portion of the fusionprotein may be contributed by another cytokine receptor, such as IL-10R,IL-13R, IL-17R, IL-10RB (CRF24), or by a non-native and/or an unrelatedsecretory signal peptide that facilitates secretion of the fusionprotein.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a ZcytoR14 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides, antigenicpeptides, epitopes, and polypeptides of the present invention are usefulto raise antibodies that bind with the polypeptides described herein, aswell as to identify and screen anti-ZcytoR14 monoclonal antibodies thatare neutralizing, and that may bind, block, inhibit, reduce, antagonizeor neutralize the activity of IL-17F and EL-17A (individually ortogether). Such neutralizing monoclonal antibodies of the presentinvention can bind to an ZcytoR14 antigenic epitope. Hopp/Woodshydrophilicity profiles can be used to determine regions that have themost antigenic potential within SEQ ID NO:2 or 4 (Hopp et al., Proc.Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. In ZcytoR14 these regionscan be determined by one of skill in the art. Moreover, ZcytoR14antigenic epitopes within SEQ ID NO:2 or 4 as predicted by aJameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc.,Madison, Wis.) serve as preferred antigenic epitpoes, and can bedetermined by one of skill in the art. The results of this analysisindicated that the following amino acid sequences of SEQ ID NO:2 wouldprovide suitable antigenic peptides: amino acids 26 to 33 (“antigenicpeptide 1”), amino acids 41 to 46 (“antigenic peptide 2”), 74 to 81(“antigenic peptide 3”), amino acids 95 to 105 (“antigenic peptide 4”),amino acids 109 to 119 (“antigenic peptide 5”), amino acids 95 to 119(“antigenic peptide 6”), amino acids 178 to 185 (“antigenic peptide 7”),amino acids 200 to 206 (“antigenic peptide 8”), amino acids 231 to 238(“antigenic peptide 9”), amino acids 231 to 241 (“antigenic peptide10”), amino acids 264 to 270 (“antigenic peptide 11”), amino acids 274to 281 (“antigenic peptide 12”), amino acids 317 to 324 (“antigenicpeptide 13”), amino acids 357 to 363 (“antigenic peptide 14”), aminoacids 384 to 392 (“antigenic peptide 15”), amino acids 398 to 411(“antigenic peptide 16”), amino acids 405 to 411 (“antigenic peptide17”), amino acids 423 to 429 (“antigenic peptide 18”), and amino acids434 to 439 (“antigenic peptide 19”). The present invention contemplatesthe use of any one of antigenic peptides 1 to 19 to generate antibodiesto Zcytor14. The present invention also contemplates polypeptidescomprising at least one of antigenic peptides 1 to 19.

In preferred embodiments, antigenic epitopes to which neutralizingantibodies of the present invention bind would contain residues of SEQID NO:2 (and corresponding residues of SEQ ID NO:3) or SEQ ID NO:5 thatare important to ligand-receptor binding, for example, with ZcytoR14 andIL-17A or IL-17F (individually or together).

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of an amino acid sequence disclosed herein.Such epitope-bearing peptides and polypeptides can be produced byfragmenting a ZcytoR14 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol.7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

For any ZcytoR14 polypeptide, including variants and fusion proteins,one of ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise ZcytoR14 variants based upon thenucleotide and amino acid sequences described herein.

E) Production of ZcytoR14 Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides; soluble monomeric, homodimeric, heterodimeric andmultimeric receptors; full-length receptors; receptor fragments (e.g.ligand-binding fragments and antigenic epitopes), functional fragments,and fusion proteins, can be produced in recombinant host cells followingconventional techniques. To express a ZcytoR14 gene, a nucleic acidmolecule encoding the polypeptide must be operably linked to regulatorysequences that control transcriptional expression in an expressionvector and then, introduced into a host cell. In addition totranscriptional regulatory sequences, such as promoters and enhancers,expression vectors can include translational regulatory sequences and amarker gene which is suitable for selection of cells that carry theexpression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, an ZcytoR14 expression vector maycomprise a ZcytoR14 gene and a secretory sequence derived from anysecreted gene.

ZcytoR14 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from mammalian viral sources, for example,adenovirus, bovine papilloma virus, simian virus, or the like, in whichthe regulatory signals are associated with a particular gene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, forexample, actin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control ZcytoR14 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

In certain embodiments, a DNA sequence encoding a ZcytoR14 solublereceptor polypeptide, or a fragment of ZcytoR14 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers. Multiple components of asoluble receptor complex can be co-transfected on individual expressionvectors or be contained in a single expression vector. Such techniquesof expressing multiple components of protein complexes are well known inthe art.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase (DHFR), which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

ZcytoR14 polypeptides can also be produced by cultured mammalian cellsusing a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, retroviruses, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see Becker et al., Meth. CellBiol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44(1997)). Advantages of the adenovirus system include the accommodationof relatively large DNA inserts, the ability to grow to high-titer, theability to infect a broad range of mammalian cell types, and flexibilitythat allows use with a large number of available vectors containingdifferent promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

ZcytoR14 can also be expressed in other higher eukaryotic cells, such asavian, fungal, insect, yeast, or plant cells. The baculovirus systemprovides an efficient means to introduce cloned ZcytoR14 genes intoinsect cells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the ZcytoR14 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed ZcytoR14 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a ZcytoR14 gene is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native ZcytoR14 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native ZcytoR14 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2−5×10⁵ cells to adensity of 1−2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillernondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRB1). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, ZcytoR14 genes can be expressed in prokaryotic hostcells. Suitable promoters that can be used to express ZcytoR14polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a ZcytoR14 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:2 or 5. As an illustration, polypeptides can comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of ofSEQ ID NO:2 or 5. Within certain embodiments of the invention, thepolypeptides comprise 20, 30, 40, 50, 100, or more contiguous residuesof these amino acid sequences. Nucleic acid molecules encoding suchpeptides and polypeptides are useful as polymerase chain reactionprimers and probes.

Moreover, ZcytoR14 polypeptides and fragments thereof can be expressedas monomers, homodimers, heterodimers, or multimers within highereukaryotic cells. Such cells can be used to produce ZcytoR14 monomeric,homodimeric, heterodimeric and multimeric receptor polypeptides thatcomprise at least one ZcytoR14 polypeptide (“ZcytoR14-comprisingreceptors” or “ZcytoR14-comprising receptor polypeptides”), or can beused as assay cells in screening systems. Within one aspect of thepresent invention, a polypeptide of the present invention comprising theZcytoR14 extracellular domain is produced by a cultured cell, and thecell is used to screen for ligands for the receptor, including thenatural ligand, IL-17F, as well as IL-17A, or even agonists andantagonists of the natural ligand. To sunamarize this approach, a cDNAor gene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems. Each component of the monomeric,homodimeric, heterodimeric and multimeric receptor complex can beexpressed in the same cell. Moreover, the components of the monomeric,homodimeric, heterodimeric and multimeric receptor complex can also befused to a transmembrane domain or other membrane fusion moiety to allowcomplex assembly and screening of transfectants as described above.

To assay the IL-17A and IL-17F antagonist polyepeptides and antibodiesof the present invention, mammalian cells suitable for use in expressingZcytoR14-comprising receptors or other receptors known to bind IL-17A orIL-17F (e.g., cells expressing IL-17R) and transducing areceptor-mediated signal include cells that express other receptorsubunits that may form a functional complex with ZcytoR14. It is alsopreferred to use a cell from the same species as the receptor to beexpressed. Within a preferred embodiment, the cell is dependent upon anexogenously supplied hematopoietic growth factor for its proliferation.Preferred cell lines of this type are the human TF-1 cell line (ATCCnumber CRL-2003) and the AML-193 cell line (ATCC number CRL-9589), whichare GM-CSF-dependent human leukemic cell lines and BaF3 (Palacios andSteinmetz, Cell 41: 727-734, (1985)) which is an IL-3 dependent murinepre-B cell line. Other cell lines include BHK, COS-1 and CHO cells.Suitable host cells can be engineered to produce the necessary receptorsubunits or other cellular component needed for the desired cellularresponse. This approach is advantageous because cell lines can beengineered to express receptor subunits from any species, therebyovercoming potential limitations arising from species specificity.Species orthologs of the human receptor cDNA can be cloned and usedwithin cell lines from the same species, such as a mouse cDNA in theBaF3 cell line. Cell lines that are dependent upon one hematopoieticgrowth factor, such as GM-CSF or IL-3, can thus be engineered to becomedependent upon another cytokine that acts through the ZcytoR14 receptor,such as IL-17F or IL-17A.

Cells expressing functional receptor are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the metabolic breakdown of3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assayformat uses cells that are further engineered to express a reportergene. The reporter gene is linked to a promoter element that isresponsive to the receptor-linked pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, or SRE. See, e.g.,Shaw et al., Cell 56:563-572, (1989). A preferred such reporter gene isa luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, (1987)).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:29094-29101, (1994); Schenborn and Goiffin, Promega _(—) Notes41:11, 1993). Luciferase activity assay kits are commercially availablefrom, for example, Promega Corp., Madison, Wis. Target cell lines ofthis type can be used to screen libraries of chemicals, cell-conditionedculture media, fungal broths, soil samples, water samples, and the like.For example, a bank of cell-conditioned media samples can be assayed ona target cell to identify cells that produce ligand. Positive cells arethen used to produce a cDNA library in a mammalian expression vector,which is divided into pools, transfected into host cells, and expressed.Media samples from the transfected cells are then assayed, withsubsequent division of pools, re-transfection, subculturing, andre-assay of positive cells to isolate a cloned cDNA encoding the ligand.

An additional screening approach provided by the present inventionincludes the use of hybrid receptor polypeptides. These hybridpolypeptides fall into two general classes. Within the first class, theintracellular domain of ZcytoR14, is joined to the ligand-binding domainof a second receptor. A second class of hybrid receptor polypeptidescomprise the extracellular (ligand-binding) domain of ZcytoR14 (SEQ IDNO:3) with an intracellular domain of a second receptor, preferably ahematopoietic cytokine receptor, and a transmembrane domain. HybridZcytoR14 monomers, homodimers, heterodimers and multimers of the presentinvention receptors of this second class are expressed in cells known tobe capable of responding to signals transduced by the second receptor.Together, these two classes of hybrid receptors enable theidentification of a responsive cell type for the development of an assayfor detecting IL-17F or IL-17A. Moreover, such cells can be used in thepresence of IL-17F or IL-17A to assay the soluble receptor antagonistsof the present invention in a competition-type assay. In such assay, adecrease in the proliferation or signal transduction activity of IL-17For IL-17A in the presence of a soluble receptor of the present inventiondemonstrates antagonistic activity. Moreover ZcytoR14-soluble receptorbinding assays, an cell-based assays, can also be used to assess whethera soluble receptor binds, blocks, inhibits, reduces, antagonizes orneutralizes IL-17F or IL-17A activity.

F) Production of ZcytoR14 Fusion Proteins and Conjugates

One general class of ZcytoR14 analogs are variants having an amino acidsequence that is a mutation of the amino acid sequence disclosed herein.Another general class of ZcytoR14 analogs is provided by anti-idiotypeantibodies, and fragments thereof, as described below. Moreover,recombinant antibodies comprising anti-idiotype variable domains can beused as analogs (see, for example, Monfardini et al., Proc. Assoc. Am.Physicians 108:420 (1996)). Since the variable domains of anti-idiotypeZcytoR14 antibodies mimic ZcytoR14, these domains can provide ZcytoR14binding activity. Methods of producing anti-idiotypic catalyticantibodies are known to those of skill in the art (see, for example,Joron et al., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et al.,Appl. Biochem. Biotechnol. 47:229 (1994), and Avalle et al., Ann. N YAcad. Sci. 864:118 (1998)).

Another approach to identifying ZcytoR14 analogs is provided by the useof combinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

ZcytoR14 polypeptides have both in vivo and in vitro uses. As anillustration, a soluble form of ZcytoR14 can be added to cell culturemedium to inhibit the effects of the ZcytoR14 ligand (i.e. IL-17F,IL-17A or both) produced by the cultured cells.

Fusion proteins of ZcytoR14 can be used to express ZcytoR14 in arecombinant host, and to isolate the produced ZcytoR14. As describedbelow, particular ZcytoR14 fusion proteins also have uses in diagnosisand therapy. One type of fusion protein comprises a peptide that guidesa ZcytoR14 polypeptide from a recombinant host cell. To direct aZcytoR14 polypeptide into the secretory pathway of a eukaryotic hostcell, a secretory signal sequence (also known as a signal peptide, aleader sequence, prepro sequence or pre sequence) is provided in theZcytoR14 expression vector. While the secretory signal sequence may bederived from ZcytoR14, a suitable signal sequence may also be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to a ZcytoR14-encoding sequence suchthat the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the nucleotide sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleotide sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830).

Although the secretory signal sequence of ZcytoR14 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of ZcytoR14 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone ac-factor (encoded by the MFα1 gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al., “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

ZcytoR14 soluble receptor polypeptides can be prepared by expressing atruncated DNA encoding the extracellular domain, for example, apolypeptide which contains SEQ ID NO:3, or the corresponding region of anon-human receptor. It is preferred that the extracellular domainpolypeptides be prepared in a form substantially free of transmembraneand intracellular polypeptide segments. To direct the export of thereceptor domain from the host cell, the receptor DNA is linked to asecond DNA segment encoding a secretory peptide, such as a t-PAsecretory peptide. To facilitate purification of the secreted receptordomain, a C-terminal extension, such as a poly-histidine tag, substanceP, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210, (1988);available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide. Moreover,ZcytoR14 antigenic epitopes from the extracellular cytokine bindingdomains are also prepared as described above.

In an alternative approach, a receptor extracellular domain of ZcytoR14or other cytokine receptor component can be expressed as a fusion withimmunoglobulin heavy chain constant regions, typically an F_(C)fragment, which contains two constant region domains and a hinge regionbut lacks the variable region (See, Sledziewski, AZ et al., U.S. Pat.No. 6,018,026 and 5,750,375). The soluble ZcytoR14 polypeptides of thepresent invention include such fusions. One such fusion is shown in SEQID NO:64. Such fusions are typically secreted as multimeric moleculeswherein the Fc portions are disulfide bonded to each other and tworeceptor polypeptides are arrayed in closed proximity to each other.Fusions of this type can be used to affinity purify the cognate ligandfrom solution, as an in vitro assay tool, to block, inhibit or reducesignals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a ZcytoR14-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. The chimeras maybe used in vivo to regulate inflammatory responses including acute phaseresponses such as serum amyloid A (SAA), C-reactive protein (CRP), andthe like. Chimeras with high binding affinity are administeredparenterally (e.g., by intramuscular, subcutaneous or intravenousinjection). Circulating molecules bind ligand and are cleared fromcirculation by normal physiological processes. For use in assays, thechimeras are bound to a support via the F_(C) region and used in anELISA format.

To assist in isolating anti-ZcytoR14 and binding partners of the presentinvention, an assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulfhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding. Alternatively, ligand/receptor binding can beanalyzed using SELDI(TM) technology (Ciphergen, Inc., Palo Alto,Calif.). Moreover, BIACORE technology, described above, can be used tobe used in competition experiments to determine if different momnoclonalantibodies bind the same or different epitopes on the ZcytoR14polypeptide, and as such, be used to aid in epitope mapping ofneutralizing antibodies of the present invention that bind, block,inhibit, reduce, antagonize or neutralize IL-17F or both IL-17A andIL-17F.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a soluble ZcytoR14 receptor can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains, e.g., IgGΓ1, andthe human κ light chain. Immunoglobulin-soluble ZcytoR14 fusions can beexpressed in genetically engineered cells to produce a variety ofmultimeric ZcytoR14 receptor analogs. Auxiliary domains can be fused tosoluble ZcytoR14 receptor to target them to specific cells, tissues, ormacromolecules (e.g., collagen, or cells expressing the ZcytoR14ligands, IL-17F or IL-17A). A ZcytoR14 polypeptide can be fused to twoor more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9, 1996.

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, ZcytoR14can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a ZcytoR14fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2nd Edition, Gloverand Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolylistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

Another form of fusion protein comprises a ZcytoR14 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(C) fragment,which contains two or three constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:9). In this fusion protein, an illustrative Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a ZcytoR14 fusion protein that comprises a ZcytoR14 moietyand a human Fc fragment, wherein the C-terminus of the ZcytoR14 moietyis attached to the N-terminus of the Fc fragment via a peptide linker,such as a peptide comprising the amino acid sequence of SEQ ID NO:2 orSEQ ID NO:5. The ZcytoR14 moiety can be a ZcytoR14 molecule or afragment thereof. For example, a fusion protein can comprise the aminoacid of SEQ ID NO:3 and an Fc fragment (e.g., a human Fc fragment) (SEQID NO:64).

In another variation, a ZcytoR14 fusion protein comprises an IgGsequence, a ZcytoR14 moiety covalently joined to the aminoterminal endof the IgG sequence, and a signal peptide that is covalently joined tothe aminoterminal of the ZcytoR14 moiety, wherein the IgG sequenceconsists of the following elements in the following order: a hingeregion, a CH₂ domain, and a CH₃ domain. Accordingly, the IgG sequencelacks a CH₁ domain. The ZcytoR14 moiety displays a ZcytoR14 activity, asdescribed herein, such as the ability to bind with a ZcytoR14 ligand.This general approach to producing fusion proteins that comprise bothantibody and nonantibody portions has been described by LaRochelle etal., EP 742830 (WO 95/21258).

Fusion proteins comprising a ZcytoR14 moiety and an Fc moiety can beused, for example, as an in vitro assay tool. For example, the presenceof a ZcytoR14 ligand in a biological sample can be detected using aZcytoR14-immunoglobulin fusion protein, in which the ZcytoR14 moiety isused to bind the ligand, and a macromolecule, such as Protein A oranti-Fc antibody, is used to bind the fusion protein to a solid support.Such systems can be used to identify agonists and antagonists thatinterfere with the binding of a ZcytoR14 ligands, e.g., IL-17F or bothIL-17A and EL-17F, to their receptor.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a ZcytoR14 fragment that containsa ZcytoR14 extracellular domain. Such molecules can be used to targetparticular tissues for the benefit of ZcytoR14 binding activity.

The present invention further provides a variety of other polypeptidefusions. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between ZcytoR14 of the present invention withthe functionally equivalent domain(s) from another member of thecytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of ZcytoR14 fusion analogs.A ZcytoR14 polypeptide can be fused to two or more moieties or domains,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, for example, Tuan et al., ConnectiveTissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

ZcytoR14 binding domains can be further characterized by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of ZcytoR14 ligand agonists. See, for example, de Voset al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

The present invention also contemplates chemically modified ZcytoR14compositions, in which a ZcytoR14 polypeptide is linked with a polymer.Illustrative ZcytoR14 polypeptides are soluble polypeptides that lack afunctional transmembrane domain, such as a polypeptide consisting ofamino acid residues SEQ ID NO:3. Typically, the polymer is water solubleso that the ZcytoR14 conjugate does not precipitate in an aqueousenvironment, such as a physiological environment. An example of asuitable polymer is one that has been modified to have a single reactivegroup, such as an active ester for acylation, or an aldehyde foralkylation. In this way, the degree of polymerization can be controlled.An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce ZcytoR14 conjugates.

ZcytoR14 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A ZcytoR14conjugate can also comprise a mixture of such water-soluble polymers.

One example of a ZcytoR14 conjugate comprises a ZcytoR14 moiety and apolyalkyl oxide moiety attached to the N-terminus of the ZcytoR14moiety. PEG is one suitable polyalkyl oxide. As an illustration,ZcytoR14 can be modified with PEG, a process known as “PEGylation.”PEGylation of ZcytoR14 can be carried out by any of the PEGylationreactions known in the art (see, for example, EP 0 154 316, Delgado etal., Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992),Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis etal., Int J Hematol 68:1 (1998)). For example, PEGylation can beperformed by an acylation reaction or by an alkylation reaction with areactive polyethylene glycol molecule. In an alternative approach,ZcytoR14 conjugates are formed by condensing activated PEG, in which aterminal hydroxy or amino group of PEG has been replaced by an activatedlinker (see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a ZcytoR14 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween ZcytoR14 and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated ZcytoR14 byacylation will typically comprise the steps of (a) reacting a ZcytoR14polypeptide with PEG (such as a reactive ester of an aldehyde derivativeof PEG) under conditions whereby one or more PEG groups attach toZcytoR14, and (b) obtaining the reaction product(s). Generally, theoptimal reaction conditions for acylation reactions will be determinedbased upon known parameters and desired results. For example, the largerthe ratio of PEG:ZcytoR14, the greater the percentage of polyPEGylatedZcytoR14 product.

The product of PEGylation by acylation is typically a polyPEGylatedZcytoR14 product, wherein the lysine E-amino groups are PEGylated via anacyl linking group. An example of a connecting linkage is an amide.Typically, the resulting ZcytoR14 will be at least 95% mono-, di-, ortri-pegylated, although some species with higher degrees of PEGylationmay be formed depending upon the reaction conditions. PEGylated speciescan be separated from unconjugated ZcytoR14 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with ZcytoR14 in the presence of a reducing agent. PEGgroups can be attached to the polypeptide via a —CH₂—NH group.

Moreover, anti-ZcytoR14 antibodies or antibody fragments of the presentinvention can be PEGylated using methods in the art and describedherein.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the a-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of ZcytoR14monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer ZcytoR14 conjugate molecule can comprise the steps of: (a)reacting a ZcytoR14 polypeptide with a reactive PEG under reductivealkylation conditions at a pH suitable to permit selective modificationof the α-amino group at the amino terminus of the ZcytoR14, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and able to reduce onlythe Schiff base formed in the initial process of reductive alkylation.Illustrative reducing agents include sodium borohydride, sodiumcyanoborohydride, dimethylamine borane, trimethylamine borane, andpyridine borane.

For a substantially homogenous population of monopolymer ZcytoR14conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of ZcytoR14. Such reaction conditions generally providefor pKa differences between the lysine amino groups and the α-aminogroup at the N-terminus. The pH also affects the ratio of polymer toprotein to be used. In general, if the pH is lower, a larger excess ofpolymer to protein will be desired because the less reactive theN-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer:ZcytoR14 need not be aslarge because more reactive groups are available. Typically, the pH willfall within the range of 3 to 9, or 3 to 6. This method can be employedfor making ZcytoR14-comprising homodimeric, heterodimeric or multimericsoluble receptor conjugates.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to ZcytoR14 will generallybe in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to ZcytoR14 will be 1:1 to 20:1 forpolyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)). This method can beemployed for making ZcytoR14-comprising homodimeric, heterodimeric ormultimeric soluble receptor conjugates.

The present invention contemplates compositions comprising a peptide orpolypeptide, such as a soluble receptor or antibody described herein.Such compositions can further comprise a carrier. The carrier can be aconventional organic or inorganic carrier. Examples of carriers includewater, buffer solution, alcohol, propylene glycol, macrogol, sesame oil,corn oil, and the like.

G) Isolation of ZcytoR14 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95%, such as 96%, 97%, 98%, or greater than 99%purity with respect to contaminating macromolecules, particularly otherproteins and nucleic acids, and free of infectious and pyrogenic agents.The polypeptides of the present invention may also be purified to apharmaceutically pure state, which is greater than 99.9% pure. Incertain preparations, purified polypeptide is substantially free ofother polypeptides, particularly other polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of ZcytoR14 purified from natural sources (e.g.,human tissue sources), synthetic ZcytoR14 polypeptides, and recombinantZcytoR14 polypeptides and fusion ZcytoR14 polypeptides purified fromrecombinant host cells. In general, ammonium sulfate precipitation andacid or chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are suitable. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in ZcytoR14 isolation and purification can bedevised by those of skill in the art. For example, anti-ZcytoR14antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Moreover, theligand-binding properties of ZcytoR14 extracellular domain can beexploited for purification, for example, of ZcytoR14-comprising solublereceptors; for example, by using affinity chromatography wherein IL-17Fligand is bound to a column and the ZcytoR14-comprising receptor isbound and subsequently eluted using standard chromatography methods.

ZcytoR14 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. ZcytoR14 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

H) Production of Antibodies to ZcytoR14 Proteins

Antibodies to ZcytoR14 can be obtained, for example, using the productof a ZcytoR14 expression vector or ZcytoR14 isolated from a naturalsource as an antigen. Particularly useful anti-ZcytoR14 antibodies “bindspecifically” with ZcytoR14. Antibodies are considered to bespecifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to ZcytoR14 with athreshold level of binding activity, and (2) antibodies do notsignificantly cross-react with polypeptides related to ZcytoR14.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a ZcytoR14 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect ZcytoR14, but notpresently known polypeptides using a standard Western blot analysis.Examples of known related polypeptides include known cytokine receptors.

Anti-ZcytoR14 antibodies can be produced using antigenic ZcytoR14epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or another amino acid sequencedisclosed herein. However, peptides or polypeptides comprising a largerportion of an amino acid sequence of the invention, containing from 30to 50 amino acids, or any length up to and including the entire aminoacid sequence of a polypeptide of the invention, also are useful forinducing antibodies that bind with ZcytoR14. It is desirable that theamino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues aretypically avoided). Moreover, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

As an illustration, potential antigenic sites in ZcytoR14 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Gamier-Robson, Gamier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins;aα region threshold=103; β region threshold=105; Garnier-Robsonparameters: α and β decision constants=0). In the sixth subroutine,flexibility parameters and hydropathy/solvent accessibility factors werecombined to determine a surface contour value, designated as the“antigenic index.” Finally, a peak broadening function was applied tothe antigenic index, which broadens major surface peaks by adding 20,40, 60, or 80% of the respective peak value to account for additionalfree energy derived from the mobility of surface regions relative tointerior regions. This calculation was not applied, however, to anymajor peak that resides in a helical region, since helical regions tendto be less flexible. Hopp/Woods hydrophilicity profiles can be used todetermine regions that have the most antigenic potential within SEQ IDNO:3 (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J.Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering11:153-169, 1998). The profile is based on a sliding six-residue window.Buried G, S, and T residues and exposed H, Y, and W residues wereignored. Moreover, ZcytoR14 antigenic epitopes within SEQ ID NO:3 aspredicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean program(DNASTAR, Inc., Madison, Wis.) serve as preferred antigenic epitopes,and can be determined by one of skill in the art. Such antigenicepitopes include (1) amino acid residue 73 to amino acid residue 82 ofSEQ ID NO:3; (2) amino acid residue 95 to amino acid residue 104 of SEQID NO:3; (3) amino acid residue 111 to amino acid residue 119 of SEQ IDNO:3; (4) amino acid residue 179 to amino acid residue 186 of SEQ IDNO:3; (5) amino acid residue 200 to amino acid residue 205 of SEQ IDNO:3; (6) amino acid residue 229 to amino acid residue 236 of SEQ IDNO:3; (7) amino acid residue 264 to amino acid residue 268 of SEQ IDNO:3; and (8) amino acid residue 275 to amino acid residue 281 of SEQ IDNO:3. The present invention contemplates the use of any one of antigenicpeptides X to Y to generate antibodies to ZcytoR14 or as a tool toscreen or identify neutralizing monoclonal antibodies of the presentinvention. The present invention also contemplates polypeptidescomprising at least one of antigenic peptides X to Y. The presentinvention contemplates the use of any antigenic peptides or epitopesdescribed herein to generate antibodies to ZcytoR14, as well as toidentify and screen anti-ZcytoR14 monoclonal antibodies that areneutralizing, and that may bind, block, inhibit, reduce, antagonize orneutralize the activity of IL-17F and IL-17A (individually or together).

Moreover, suitable antigens also include the ZcytoR14 polypeptidescomprising a ZcytoR14 cytokine binding, or extracellular domaindisclosed above in combination with another cytokine extracellulardomain, such as a class I or II cytokine receptor domain, such as thosethat may form soluble ZcytoR14 heterodimeric or multimeric polypeptides,and the like.

Polyclonal antibodies to recombinant ZcytoR14 protein or to ZcytoR14isolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a ZcytoR14 polypeptide canbe increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of ZcytoR14 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-ZcytoR14 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-ZcytoR14 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a ZcytoR14 gene product, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-ZcytoR14 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-ZcytoR14 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZcytoR14 polypeptide in vitro, and selecting antibody display librariesin phage or similar vectors (for instance, through use of immobilized orlabeled ZcytoR14 protein or peptide). Genes encoding polypeptides havingpotential ZcytoR14 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the ZcytoR14 sequences disclosed herein toidentify proteins which bind to ZcytoR14.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-ZcytoR14 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Moreover, anti-ZcytoR14 antibodies or antibody fragments of the presentinvention can be PEGylated using methods in the art and describedherein.

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-ZcytoR14 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-ZcytoR14 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

An anti-ZcytoR14 antibody can be conjugated with a detectable label toform an anti-ZcytoR14 immunoconjugate. Suitable detectable labelsinclude, for example, a radioisotope, a fluorescent label, achemiluminescent label, an enzyme label, a bioluminescent label orcolloidal gold. Methods of making and detecting such detectably-labeledimmunoconjugates are well-known to those of ordinary skill in the art,and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-ZcytoR14 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-ZcytoR14 immunoconjugates can be detectably labeledby coupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-ZcytoR14immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Altematively, anti-ZcytoR14 immunoconjugates can be detectably labeledby linking an anti-ZcytoR14 antibody component to an enzyme. When theanti-ZcytoR14-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-ZcytoR14 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101(1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-ZcytoR14 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for ZcytoR14 gene expression. Such kitscomprise at least one container comprising an anti-ZcytoR14 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of ZcytoR14antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that ZcytoR14 antibodies or antibody fragments areused to detect ZcytoR14 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect ZcytoR14. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

I) Use of Anti-ZcytoR14 Antibodies to Antagonize ZcytoR14 Binding toIL-17F or Both IL-17A and IL-17F

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to soluble ZcytoR14receptor polypeptides or fragments thereof, such as antigenic epitopes,and selection of antibody display libraries in phage or similar vectors(for instance, through use of immobilized or labeled soluble ZcytoR14receptor polypeptides or fragments thereof, such as antigenic epitopes).Genes encoding polypeptides having potential binding domains such assoluble ZcytoR14 receptor polypeptides or fragments thereof, such asantigenic epitopes can be obtained by screening random peptide librariesdisplayed on phage (phage display) or on bacteria, such as E. coli.Nucleotide sequences encoding the polypeptides can be obtained in anumber of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides that interact with a known target that canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409;Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No.5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptidedisplay libraries and kits for screening such libraries are availablecommercially, for instance from Clontech (Palo Alto, Calif.), InvitrogenInc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) andPharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptidedisplay libraries can be screened using the soluble ZcytoR14 receptorpolypeptides or fragments thereof, such as antigenic epitope polypeptidesequences disclosed herein to identify proteins which bind toZcytoR14-comprising receptor polypeptides. These “binding polypeptides,”which interact with soluble ZcytoR14-comprising receptor polypeptides,can be used for tagging cells; for isolating homolog polypeptides byaffinity purification; they can be directly or indirectly conjugated todrugs, toxins, radionuclides and the like. These binding polypeptidescan also be used in analytical methods such as for screening expressionlibraries and neutralizing activity, e.g., for binding, blocking,inhibiting, reducing, antagonizing or neutralizing interaction betweenIL-17A and IL-17F (individually or together) and ZcytoR14, or viralbinding to a receptor. The binding polypeptides can also be used fordiagnostic assays for determining circulating levels of solubleZcytoR14-comprising receptor polypeptides; for detecting or quantitatingsoluble or non-soluble ZcytoR14-comprising receptors as marker ofunderlying pathology or disease. These binding polypeptides can also actas “antagonists” to block or inhibit soluble or membrane-bound ZcytoR14monomeric receptor or ZcytoR14 homodimeric, heterodimeric or multimericpolypeptide binding (e.g. to ligand) and signal transduction in vitroand in vivo. Again, these binding polypeptides serve as anti-ZcytoR14monomeric receptor or anti-ZcytoR14 homodimeric, heterodimeric ormultimeric polypeptides and are useful for inhibiting IL-17F or bothIL-17A and IL-17F activity, as well as receptor activity orprotein-binding. Antibodies raised to the natural receptor complexes ofthe present invention, and ZcytoR14-epitope-binding antibodies, andanti-ZcytoR14 neutralizing monoclonal antibodies may be preferredembodiments, as they may act more specifically against the ZcytoR14 andcan inhibit IL-17F or both IL-17A and IL-17F. Moreover, the antagonisticand binding activity of the antibodies of the present invention can beassayed in an IL-17A or IL-17F proliferation, signal trap, luciferase orbinding assays in the presence of IL-17A or IL-17F respectively, andZcytoR14-comprising soluble receptors, and other biological orbiochemical assays described herein.

Antibodies to soluble ZcytoR14 receptor polypeptides (e.g., antibodiesto SEQ ID NO:3) or fragments thereof, such as antigenic epitopes may beused for inhibiting the inflammatory effects of IL-17A, IL-17F, or bothIL-17A and IL-17F in vivo, for theraputic use against inflammation andinflammatory dieases such as psoriasis, psoriatic arthritis, rheumatoidarthritis, endotoxemia, inflammatory bowel disease (IBD), colitis,asthma, allograft rejection, immune mediated renal diseases,hepatobiliary diseases, multiple sclerosis, atherosclerosis, promotionof tumor growth, or degenerative joint disease and other inflammatoryconditions disclosed herein; tagging cells that express ZcytoR14receptors; for isolating soluble ZcytoR14-comprising receptorpolypeptides by affinity purification; for diagnostic assays fordetermining circulating levels of soluble ZcytoR14-comprising receptorpolypeptides; for detecting or quantitating soluble ZcytoR14-comprisingreceptors as marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies that can act as IL-17F or IL-17Aagonists; and as neutralizing antibodies or as antagonists to bind,block, inhibit, reduce, or antagonize ZcytoR14 receptor function, or tobind, block, inhibit, reduce, antagonize or neutralize IL-17F and/orIL-17A activity (either individually or together) in vitro and in vivo.Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, biotin, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like; indirect tagsor labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies herein mayalso be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to solubleZcytoR14-comprising receptor polypeptides, or fragments thereof may beused in vitro to detect denatured or non-denatured ZcytoR14-comprisingreceptor polypeptides or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Antibodies to soluble ZcytoR14 receptor or soluble ZcytoR14 homodimeric,heterodimeric or multimeric receptor polypeptides are useful for taggingcells that express the corresponding receptors and assaying theirexpression levels, for affinity purification, within diagnostic assaysfor deterrnining circulating levels of receptor polypeptides, analyticalmethods employing fluorescence-activated cell sorting. Moreover,divalent antibodies, and anti-idiotypic antibodies may be used asagonists to mimic the effect of the ZcytoR14 ligand, IL-17F or IL-17A.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize soluble ZcytoR14 receptor orsoluble ZcytoR14 homodimeric, heterodimeric or multimeric receptorpolypeptides can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (i.e., aZcytoR14-comprising soluble or membrane-bound receptor). Morespecifically, antibodies to soluble ZcytoR14-comprising receptorpolypeptides, or bioactive fragments or portions thereof, can be coupledto detectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express the ZcytoR14-comprising receptorsuch as ZcytoR14-expressing cancers.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind ZcytoR14-comprising receptor polypeptides, suchas “binding polypeptides,” (including binding peptides disclosed above),antibodies, or bioactive fragments or portions thereof. Suitabledetectable molecules include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent markers, chemiluminescent markers,magnetic particles and the like. Suitable cytotoxic molecules may bedirectly or indirectly attached to the polypeptide or antibody, andinclude bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Binding polypeptidesor antibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the binding polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, ZcytoR14 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, spleen, pancreatic, blood,lymphoid, colon, and bone marrow cancers), if the bindingpolypeptide-cytokine or anti-ZcytoR14 receptor antibody targets thehyperproliferative cell (See, generally, Hornick et al., Blood89:4437-47, 1997). The described fusion proteins enable targeting of acytokine to a desired site of action, thereby providing an elevatedlocal concentration of cytokine. Suitable anti-ZcytoR14 monomer,homodimer, heterodimer or multimer antibodies target an undesirable cellor tissue (i.e., a tumor or a leukemia), and the fused cytokine mediatesimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

Alternatively, ZcytoR14 receptor binding polypeptides or antibody fusionproteins described herein can be used for enhancing in vivo killing oftarget tissues by directly stimulating a ZcytoR14 receptor-modulatedapoptotic pathway, resulting in cell death of hyperproliferative cellsexpressing ZcytoR14-comprising receptors.

J) Therapeutic Uses of Polypeptides Having ZcytoR14 Activity orAntibodies to ZcytoR14

Amino acid sequences having soluble ZcytoR14 activity can be used tomodulate the immune system by binding ZcytoR14 ligands IL-17A and IL-17F(either singly or together), and thus, preventing the binding ofZcytoR14 ligand with endogenous ZcytoR14 receptor. ZcytoR14 antagonists,such as soluble ZcytoR14 or anti-ZcytoR14 antibodies, can also be usedto modulate the immune system by inhibiting the binding of ZcytoR14ligand with the endogenous ZcytoR14 receptor. Accordingly, the presentinvention includes the use of proteins, polypeptides, and peptideshaving ZcytoR14 activity (such as soluble ZcytoR14 polypeptides,ZcytoR14 polypeptide fragments, ZcytoR14 analogs (e.g., anti-ZcytoR14anti-idiotype antibodies), and ZcytoR14 fusion proteins) to a subjectwhich lacks an adequate amount of this polypeptide, or which produces anexcess of ZcytoR14 ligand. ZcytoR14 antagonists (e.g., anti-ZcytoR14antibodies) can be also used to treat a subject which produces an excessof either ZcytoR14 ligand or ZcytoR14. Suitable subjects includemammals, such as humans. For example, such ZcytoR14 polypeptides andanti-ZcytoR14 antibodies are useful in binding, blocking, inhibiting,reducing, antagonizing or neutralizing IL-17A and IL-17F (either singlyor together), in the treatment of inflammation and inflammatory dieasessuch as psoriasis, psoriatic arthritis, rheumatoid arthritis,endotoxemia, inflammatory bowel disease (IBD), colitis, asthma,allograft rejection, immune mediated renal diseases, hepatobiliarydiseases, multiple sclerosis, atherosclerosis, promotion of tumorgrowth, or degenerative joint disease and other inflammatory conditionsdisclosed herein.

Within preferred embodiments, the soluble receptor form of ZcytoR14, SEQID NO:3) is a monomer, homodimer, heterodimer, or multimer that bindsto, blocks, inhibits, reduces, antagonizes or neutralizes IL-17F andIL-17A (individually or together) in vivo. Antibodies and bindingpolypeptides to such ZcytoR14 monomer, homodimer, heterodimer, ormultimers also serve as antagonists of ZcytoR14 activity, and as IL-17Aand IL-17F antagonists (singly or together), as described herein.

In addition, we have described herein that both polyclonal andmonoclonal neutralizing anti-IL-17F antibodies bind to, block, inhibit,reduce, antagonize or neutralize IL-17F and IL-17A activity in cellbased neutralization assays. Analysis of the tissue distribution of themRNA corresponding ZcytoR14 cDNA showed that rnRNA the ZcytoR14 gene isstrongly expressed in thyroid, adrenal gland, prostate, and livertissues, and expressed to a lesser extent in heart, small intestine,stomach, and trachea tissues. In particular, ZcytoR14 is consistentlyexpressed in non-T cell peripheral blood cell lines, includingmonocytes, B-cells, and cells of the myeloid lineage. Also, ZcytoR14mRNA is reliably expressed in cell lines derived from skin. Other celllines that express ZcytoR14 are all 5 of the large intestine cell linesthat were present on the array. In contrast, there is little or noexpression in brain, placenta, lung, skeletal muscle, kidney, pancreas,spleen, thymus, testis, ovary, colon, peripheral blood leukocytes,spinal cord, lymph node, and bone marrow. The ligand to which ZcytoR14binds (IL-17F and/or IL-17A) is implicated in inducing inflammatoryresponse and contributing to inflammatory diseases, primarily via itsability to enhance production of inflammatory mediators, includingIL-1b, IL-6 and TNF-a, as well as those mediators that are involved inthe proliferation, maturation and chemotaxis of neutrophils (reviewed inWitowski et al. Cell. Mol. Life Sci. 61:567-579 [20041).

Thus, particular embodiments of the present invention are directedtoward use of soluble ZcytoR14 and anti-ZcytoR14 antibodies asantagonists in inflammatory and immune diseases or conditions such aspsoriasis, psoriatic arthritis, atopic dermatitis, inflammatory skinconditions, rheumatoid arthritis, inflammatory bowel disease (IBD),Crohn's Disease, diverticulosis, asthma, pancreatitis, type I diabetes(IDDM), pancreatic cancer, pancreatitis, Graves Disease, colon andintestinal cancer, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to endotoxemia, trauma, sugery orinfection; amyloidosis; splenomegaly; graft versus host disease; andwhere inhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells), suppression ofimmune response to a pathogen or antigen, or other instances whereinhibition of IL-17F or IL-17A cytokines is desired.

Moreover, antibodies or binding polypeptides that bind ZcytoR14polypeptides described herein, and ZcytoR14 polypeptides themselves areuseful to:

1) Block, inhibit, reduce, antagonize or neutralize signaling via IL-17Aor IL-17F receptors in the treatment of acute inflammation, inflammationas a result of trauma, tissue injury, surgery, sepsis or infection, andchronic inflammatory diseases such as asthma, inflammatory bowel disease(IBD), chronic colitis, splenomegaly, rheumatoid arthritis, recurrentacute inflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response.

2) Block, inhibit, reduce, antagonize or neutralize signaling via IL-17Aor IL-17F receptors in the treatment of autoimmune diseases such asIDDM, multiple sclerosis (MS), systemic Lupus erythematosus (SLE),myasthenia gravis, rheumatoid arthritis, and IBD to prevent or inhibitsignaling in immune cells (e.g. lymphocytes, monocytes, leukocytes) viaZcytoR14. Alternatively antibodies, such as monoclonal antibodies (MAb)to ZcytoR14-comprising receptors, can also be used as an antagonist todeplete unwanted immune cells to treat autoimmune disease. Asthma,allergy and other atopic disease may be treated with an MAb against, forexample, soluble ZcytoR14 soluble receptors to inhibit the immuneresponse or to deplete offending cells. Blocking, inhibiting, reducing,or antagonizing signaling via ZcytoR14, using the polypeptides andantibodies of the present invention, may also benefit diseases of thepancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,pancreatitis, and pancreatic carcinoma may benefit. ZcytoR14 may serveas a target for MAb therapy of cancer where an antagonizing MAb inhibitscancer growth and targets immune-mediated killing. (Holliger P, andHoogenboom, H: Nature Biotech. 16: 1015-1016, 1998). Mabs to solubleZcytoR14 may also be useful to treat nephropathies such asglomerulosclerosis, membranous neuropathy, amyloidosis (which alsoaffects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

3) Agonize, enhance, increase or initiate signaling via IL-17A or IL-17Freceptors in the treatment of autoimmune diseases such as IDDM, MS, SLE,myasthenia gravis, rheumatoid arthritis, and IBD. Anti-ZcytoR14neutralizing and monoclonal antibodies may signal lymphocytes or otherimmune cells to differentiate, alter proliferation, or change productionof cytokines or cell surface proteins that ameliorate autoimmunity.Specifically, modulation of a T-helper cell response to an alternatepattern of cytokine secretion may deviate an autoimmune response toameliorate disease (Smith J A et al., J. Immunol. 160:4841-4849, 1998).Similarly, agonistic anti-soluble ZcytoR14 monomers, homodimers,heterodimers and multimer monoclonal antibodies may be used to signal,deplete and deviate immune cells involved in asthma, allergy and atopoicdisease. Signaling via ZcytoR14 may also benefit diseases of thepancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,pancreatitis, and pancreatic carcinoma may benefit. ZcytoR14 may serveas a target for MAb therapy of pancreatic cancer where a signaling MAbinhibits cancer growth and targets immune-mediated killing (Tutt, A L etal., J Immunol. 161: 3175-3185, 1998). Similarly renal cell carcinomamay be treated with monoclonal antibodies to ZcytoR14-comprising solublereceptors of the present invention.

Soluble ZcytoR14 polypeptides described herein can be used to bind,block, inhibit, reduce, antagonize or neutralize IL-17F or IL-17Aactivity, either singly or together, in the treatment of autoimmunedisease, atopic disease, NIDDM, pancreatitis and kidney dysfunction asdescribed above. A soluble form of ZcytoR14 may be used to promote anantibody response mediated by Th cells and/or to promote the productionof IL-4 or other cytokines by lymphocytes or other immune cells.

The soluble ZcytoR14comprising receptors of the present invention areuseful as antagonists of IL-17A or IL-17F cytokine. Such antagonisticeffects can be achieved by direct neutralization or binding of IL-17A orIL-17F. In addition to antagonistic uses, the soluble receptors of thepresent invention can bind IL-17F and act as carrier proteins for IL-17Aor IL-17F cytokine, in order to transport the ligand to differenttissues, organs, and cells within the body. As such, the solublereceptors of the present invention can be fused or coupled to molecules,polypeptides or chemical moieties that direct thesoluble-receptor-Ligand complex to a specific site, such as a tissue,specific immune cell, or tumor. For example, in acute infection or somecancers, benefit may result from induction of inflammation and localacute phase response proteins by the action of IL-17F. Thus, the solublereceptors of the present invention can be used to specifically directthe action of IL-17A or IL-17F. See, Cosman, D. Cytokine 5: 95-106,1993; and Fernandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.

Moreover, the soluble receptors of the present invention can be used tostabilize the IL-17F or IL-17A, to increase the bioavailability,therapeutic longevity, and/or efficacy of the Ligand by stabilizing theLigand from degradation or clearance, or by targeting the ligand to asite of action within the body. For example the naturally occurringIL-6/soluble IL-6R complex stabilizes IL-6 and can signal through thegp130 receptor. See, Cosman, D. supra., and Femandez-Botran, R. supra.Moreover, ZcytoR14 may be combined with a cognate ligand such as IL-17Fto comprise a ligand/soluble receptor complex. Such complexes may beused to stimulate responses from cells presenting a companion receptorsubunit such as, for example, pDIRS1 (IL-17ARB) or CRF24 (IL-10RB). Thecell specificity of ZcytoR14/ligand complexes may differ from that seenfor the ligand administered alone. Furthermore the complexes may havedistinct pharmacokinetic properties such as affecting half-life,dose/response and organ or tissue specificity. ZcytoR14/IL-17F orZcytoR14/lL-17A complexes thus may have agonist activity to enhance animmune response or stimulate mesangial cells or to stimulate hepaticcells. Alternatively only tissues expressing a signaling subunit theheterodimerizes with the complex may be affected analogous to theresponse to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l. Acad.Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.) “The CytokineHandbook”, 3^(rd) Ed., p. 208-209). Soluble receptor/cytokine complexesfor IL-12 and CNTF display similar activities.

Moreover Inflammation is a protective response by an organism to fendoff an invading agent. Inflammation is a cascading event that involvesmany cellular and humoral mediators. On one hand, suppression ofinflammatory responses can leave a host immunocompromised; however, ifleft unchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., psoriasis, arthritis, rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease and the like),septic shock and multiple organ failure. Importantly, these diversedisease states share common inflammatory mediators. The collectivediseases that are characterized by inflammation have a large impact onhuman morbidity and mortality. Therefore it is clear thatanti-inflammatory proteins, such as ZcytoR14, and anti-ZcytoR14antibodies, could have crucial therapeutic potential for a vast numberof human and animal diseases, from asthma and allergy to autoimmunityand septic shock.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory proteins, such as ZcytoR14 polypeptides of the presentinvention. For example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002). Oneof those mediators could be IL-17A or IL-17F, and as such a moleculethat binds or inhibits IL-17F or IL-17A activity, such as solubleZcytoR14, ZcytoR14 polypeptides, or anti ZcytoR14 antibodies or bindingpartners, could serve as a valuable therapeutic to reduce inflammationin rheumatoid arthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

One group has shown that an anti-mouse IL-17 antibody reduces symptomsin a mouse CIA-model relative to control mice, thus showing conceptuallythat soluble Zcytor14-Fc may be beneficial in treating human disease.The administration of a single mouse-IL-17-specific rat antisera reducedthe symptoms of arthritis in the animals when introducedprophylactically or after symptoms of arthritis were already present inthe model (Lubberts et al, Arthritis Rheum. 50:650-9, 2004). Therefore,Zcytor14-Fc can be used to neutralize IL-17A and/or IL-17F in thetreatment of specific human diseases such as arthritis, psoriasis,psoriatic arthritis, endotoxemia, inflammatory bowel disease (IBD),colitis, and other inflammatory conditions disclosed herein.

The administration of soluble ZcytoR14 comprising polypeptides(ZcytoR14), such as ZcytoR14-Fc4 or other ZcytoR14 soluble and fusionproteins to these CIA model mice is used to evaluate the use of solubleZcytoR14 as an antagonist to IL-17F used to ameliorate symptoms andalter the course of disease. Moreover, results showing inhibition ofIL-17F by ZcytoR14 would provide proof of concept that other IL-17Fantagonists, such as soluble ZcytoR14 or neutralizing antibodiesthereto, can also be used to ameliorate symptoms and alter the course ofdisease. Furthermore, since IL-17A and/or IL-17F induces production ofIL-1b and TNF-a, both of which are implicated in the pathogenesis andprogression of rheumatoid arthritis, the systemic or localadministration of soluble ZcytoR14 comprising polypeptides, such asZcytoR14-Fc4 or other IL-17F soluble receptors (e.g., ZcytoR14; SEQ IDNO:3) and anti-ZcytoR14 antibodies, and fusion proteins can potentiallysuppress the inflammatory response in RA. By way of example and withoutlimitation, the injection of 10-200 ug Zcytor14-Fc per mouse (one toseven times a week for up to but not limited to 4 weeks via s.c., i.p.,or i.m route of administration) can significantly reduce the diseasescore (paw score, incident of inflammation, or disease). Depending onthe initiation of Zcytor14-Fc administration (e.g. prior to or at thetime of collagen immunization, or at any time point following the secondcollagen immunization, including those time points at which the diseasehas already progressed), Zcytor14 can be efficacious in preventingrheumatoid arthritis, as well as preventing its progression. Otherpotential therapeutics include ZcytoR14 polypeptides, anti-ZcytoR14antibodies, or anti IL-17F antibodies or binding partners, and the like.

2. Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory proteins, such as ZcytoR14 polypeptides and antibodiesof the present invention, could aid in preventing and treatingendotoxemia in humans and animals. ZcytoR14 polypeptides, oranti-ZcytoR14 antibodies or binding partners, could serve as a valuabletherapeutic to reduce inflammation and pathological effects inendotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals. Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that lug injection of E. coli LPS into a C57BI/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, L-10,and G-CSF.

The administration of soluble ZcytoR14 comprising polypeptides, such asZcytoR14-Fc4 or other ZcytoR14 soluble and fusion proteins to theseLPS-induced model may be used to to evaluate the use of ZcytoR14 toameliorate symptoms and alter the course of LPS-induced disease.Moreover, results showing inhibition of IL-17F by ZcytoR14 provide proofof concept that other IL-17F antagonists, such as soluble ZcytoR14 orantibodies thereto, can also be used to ameliorate symptoms in theLPS-induced model and alter the course of disease. The model will showinduction of IL-17F by LPS injection and the potential treatment ofdisease by ZcytoR14 polypeptides. Since LPS induces the production ofpro-inflammatory factors possibly contributing to the pathology ofendotoxemia, the neutralization of IL-17F activity or otherpro-inflammatory factors by an antagonist ZcytoR14 polyepeptide can beused to reduce the symptoms of endotoxemia, such as seen in endotoxicshock. Other potential therapeutics include ZcytoR14 polypeptides,anti-ZcytoR14 antibodies, or binding partners, and the like.

3. Inflammatory Bowel Disease IBD

In the United States approximately 500,000 people suffer fromInflammatory Bowel Disease (IBD) which can affect either colon andrectum (Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. ZcytoR14polypeptides, anti-ZcytoR14 antibodies, or binding partners, could serveas a valuable therapeutic to reduce inflammation and pathologicaleffects in IBD and related diseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of soluble ZcytoR14 comprising polypeptides, such asZcytoR14-Fc4 or other ZcytoR14 soluble and fusion proteins to these TNBSor DSS models can be used to evaluate the use of soluble ZcytoR14 toameliorate symptoms and alter the course of gastrointestinal disease.Moreover, the results showing inhibition of IL-17F by ZcytoR14 provideproof of concept that other IL-17F antagonists, such as soluble ZcytoR14or antibodies thereto, can also be used to ameliorate symptoms in thecolitis/IBD models and alter the course of disease.

4. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. ZcytoR14polypeptides, anti-ZcytoR14 antibodies, or binding partners, could serveas a valuable therapeutic to reduce inflammation and pathologicaleffects in psoriasis, other inflammatory skin diseases, skin and mucosalallergies, and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

ZcytoR14 soluble receptor polypeptides and antibodies thereto may alsobe used within diagnostic systems for the detection of circulatinglevels of IL-17F or IL-17A ligand, and in the detection of IL-17Fassociated with acute phase inflammatory response. Within a relatedembodiment, antibodies or other agents that specifically bind toZcytoR14 soluble receptors of the present invention can be used todetect circulating receptor polypeptides; conversely, ZcytoR14 solublereceptors themselves can be used to detect circulating or locally-actingIL-17F or IL-17A polypeptides. Elevated or depressed levels of ligand orreceptor polypeptides may be indicative of pathological conditions,including inflammation or cancer. IL-17F is known to induce associatedacute phase inflammatory response. Moreover, detection of acute phaseproteins or molecules such as IL-17A or IL-17F can be indicative of achronic inflammatory condition in certain disease states (e.g., asthma,psoriasis, rheumatoid arthritis, colitis, IBD). Detection of suchconditions serves to aid in disease diagnosis as well as help aphysician in choosing proper therapy.

In utero administration of soluble ZcytoR14 can be used to show efficacyin vivo in disease models by reducing or eliminating the phenotypeassociated with IL-17F transgenic pups which over express IL-17F, orIL-17A transgenic pups which over express IL-17A. There are precedentsin the art for in utero treatment with antagonists such as neutralizingmonoclonal antibodies (mAbs). In one case, the development of the B-1subset of B cells was dramatically affected by treating pregnant femalemice with a mAb specific for the B cell-specific molecule, CD19 (e.g.,Krop I. Et al., Eur. J. Immunol. 26(1):23842, 1996). Krop et al.injected timed pregnant mice intraperitoneally with SOOug of ratanti-mouse CD19 mAb (or a rat isotype-matched control Ab) in PBSbeginning on day 9 of gestation, with subsequent injections every otherday until birth. Pups were also injected once with 500 ug of theseantibodies at 10 days of age. In another case, Tanaka et al., found thatin utero treatment with monoclonal antibody to IL-2 receptor beta-chaincompletely abrogates development of Thy-1+ dendritic epidermal cells.The two distinct subunits of the IL-2 receptor, i.e. the alpha-chain(IL-2R alpha) and the beta-chain (IL-2R beta), are expressed in analmost mutually exclusive fashion throughout fetal thymus ontogeny.Blocking IL-2R beta, a signal transducing component of IL-2R, byadministering a neutralizing mAb to IL-2R beta, resulted in the completeand selective disappearance of Thy-1+ skin dendritic epidermal cells.Development of any other T cell subsets was uncompromised. Thisindicated that IL-2 plays a crucial role in the development of fetal Vgamma 5+ cells and their descendants (see, Tanaka, T. et al., IntImmunol. 4(4):487-9, 1992). In addition, Schattemann GC et al., showedthat PDGF-A is required for normal murine cardiovascular developmentusing an in utero system. Several lines of evidence suggest thatplatelet-derived growth factor A chain (PDGF-A) is required for normalembryonic cardiovascular development. Introduction of anti-PDGF-Aneutralizing antibodies into mouse deciduas in utero resulted in theselective disruption of PDGF-A ligand-receptor interactions in vivo fora period of 18-24 hr and allowed assessment of whether PDGF-A isrequired for cardiovascular development and when it is required (see,Schattemann GC et al., Dev. Biol. 176(1):133-42, 1996). These results,as well as others described in the art, provide evidence thatantagonists such as neutralizing mAbs or soluble receptors can elicitstrong effects in utero. Similarly, data showing the efficacy of solublereceptors and/or neutralizing IL-17A or IL-17F with monoclonalantibodies in vivo in disease models to reduce or eliminate the skinphenotype found in IL-17A and IL-17F transgenic pups which over expressIL-17A and IL-17F respectively can be shown.

In addition to other disease models described herein, the activity ofsoluble ZcytoR14 and/or anti-ZcytoR14 antibodies on inflammatory tissuederived from human psoriatic lesions can be measured in vivo using asevere combined immune deficient (SCID) mouse model. Several mousemodels have been developed in which human cells are implanted intoimmunodeficient mice (collectively referred to as xenograft models);see, for example, Cattan A R, Douglas E, Leuk. Res. 18:513-22, 1994 andFlavell, D J, Hematological Oncology 14:67-82, 1996. As an in vivoxenograft model for psoriasis, human psoriatic skin tissue is implantedinto the SCID mouse model, and challenged with an appropriateantagonist. Moreover, other psoriasis animal models in ther art may beused to evaluate IL-17A and IL-17F antagonists, such as human psoriaticskin grafts implanted into AGR129 mouse model, and challenged with anappropriate antagonist (e.g., see, Boyman, O. et al., J. Exp. Med.Online publication #20031482, 2004, incorporated hereing by reference).Soluble ZcytoR14 or Anti-ZcytoR14 antibodies that bind, block, inhibit,reduce, antagonize or neutralize the activity of IL-17F or both IL-17Aand IL-17F are preferred antagonists, however, anti-IL-17A andanti-IL-22 antibodies (alone or in combination), soluble ZcytoR14, aswell as other IL-17A and IL-17F antagonists can be used in this model.Similarly, tissues or cells derived from human colitis, IBD, arthritis,or other inflammatory lestions can be used in the SCID model to assessthe anti-inflammatory properties of the IL-17A and IL-17F antagonistsdescribed herein.

Therapies designed to abolish, retard, or reduce inflammation usingsoluble ZcytoR14, anti-ZcytoR14 antibodies or its derivatives, agonists,conjugates or variants can be tested by administration of anti-ZcytoR14antibodies or soluble ZcytoR14 compounds to SCID mice bearing humaninflammatory tissue (e.g., psoriatic lesions and the like), or othermodels described herein. Efficacy of treatment is measured andstatistically evaluated as increased anti-inflanmmatory effect withinthe treated population over time using methods well known in the art.Some exemplary methods include, but are not limited to measuring forexample, in a psoriasis model, epidermal thickness, the number ofinflammatory cells in the upper dermis, and the grades of parakeratosis.Such methods are known in the art and described herein. For example, seeZeigler, M. et al. Lab Invest 81:1253, 2001; Zollner, T. M. et al. J.Clin. Invest. 109:671, 2002; Yamanaka, N. et al. Microbio.l Immunol.45:507, 2001; Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931, 2001;Boehncke, W. H et al. Arch. Dermatol. Res. 291:104, 1999; Boehncke, W. Het al. J. Invest. Dermatol. 116:596, 2001; Nickoloff, B. J. et al. Am.J. Pathol. 146:580, 1995; Boehncke, W. H et al. J. Cutan. Pathol. 24:1,1997; Sugai, J., M. et al. J. Dermatol. Sci. 17:85, 1998; and VilladsenL. S. et al. J. Clin. Invest. 112:1571, 2003. Inflammation may also bemonitored over time using well-known methods such as flow cytometry (orPCR) to quantitate the number of inflammatory or lesional cells presentin a sample, score (weight loss, diarrhea, rectal bleeding, colonlength) for IBD, paw disease score and inflammation score for CIA RAmodel. For example, therapeutic strategies appropriate for testing insuch a model include direct treatment using soluble ZcytoR14,anti-ZcytoR14 antibodies, other IL-17A and IL-17F antagonists (singly ortogether), or related conjugates or antagonists based on the disruptinginteraction of soluble ZcytoR14 with its ligands IL-17A and IL-17F, orfor cell-based therapies utilizing soluble ZcytoR14 or anti-ZcytoR14antibodies or its derivatives, agonists, conjugates or variants.

Moreover, Psoriasis is a chronic inflammatory skin disease that isassociated with hyperplastic epidermal keratinocytes and infiltratingmononuclear cells, including CD4+ memory T cells, neutrophils andmacrophages (Christophers, Int. Arch. Allergy Immunol., 110:199, 1996).It is currently believed that environmental antigens play a significantrole in initiating and contributing to the pathology of the disease.However, it is the loss of tolerance to self-antigens that is thought tomediate the pathology of psoriasis. Dendritic cells and CD4⁺ T cells arethought to play an important role in antigen presentation andrecognition that mediate the immune response leading to the pathology.We have recently developed a model of psoriasis based on the CD4+CD45RBtransfer model (Davenport et al., Internat. Immunopharmacol.,2:653-672). Soluble ZcytoR14 or anti-ZcytoR14 antibodies of the presentinvention are administered to the mice. Inhibition of disease scores(skin lesions, inflammatory cytokines) indicates the effectiveness ofIL-17A and IL-17F antagonists in psoriasis, e.g., anti-ZcytoR14antibodies or ZcytoR14 soluble receptors.

5. Atopic Dermatitis.

AD is a common chronic inflammatory disease that is characterized byhyperactivated cytokines of the helper T cell subset 2 (Th2). Althoughthe exact etiology of AD is unknown, multiple factors have beenimplicated, including hyperactive Th2 immune responses, autoimmunity,infection, allergens, and genetic predisposition. Key features of thedisease include xerosis (dryness of the skin), pruritus (itchiness ofthe skin), conjunctivitis, inflammatory skin lesions, Staphylococcusaureus infection, elevated blood eosinophilia, elevation of serum IgEand IgG1, and chronic dermatitis with T cell, mast cell, macrophage andeosinophil infiltration. Colonization or infection with S. aureus hasbeen recognized to exacerbate AD and perpetuate chronicity of this skindisease.

AD is often found in patients with asthma and allergic rhinitis, and isfrequently the initial manifestation of allergic disease. About 20% ofthe population in Western countries suffer from these allergic diseases,and the incidence of AD in developed countries is rising for unknownreasons. AD typically begins in childhood and can often persist throughadolescence into adulthood. Current treatments for AD include topicalcorticosteroids, oral cyclosporin A, non-corticosteroidimmunosuppressants such as tacrolimus (FK506 in ointment form), andinterferon-gamma. Despite the variety of treatments for AD, manypatients' symptoms do not improve, or they have adverse reactions tomedications, requiring the search for other, more effective therapeuticagents. The soluble ZcytoR14 polypeptides and anti-ZcytoR14 antibodiesof the present invention, including the neutralizing anti-human ZcytoR14antibodies of the present invention, can be used to neutralize IL-17Fand IL-17A in the treatment of specific human diseases such as atopticdermatitis, inflammatory skin conditions, and other inflammatoryconditions disclosed herein.

6. Asthma

IL-17 plays an important role in allergen-induced T cell activation andneutrophilic influx in the airways. The receptor for IL-17 is expressedin the airways (Yao, et al. Immunity 3:811 (1995)) and IL-17 mediatedneutrophil recruitment in allergic asthma is largely induced by thechemoattractant IL-8, GRO-α and macrophage inflammatory protein-2(MIP-2) produced by IL-17 stimulated human bronchial epithelial cells(BBECs) and human bronichial fibroblasts ( Yao, et al. J Immunol155:5483 (1995)); Molet, et al. J Allergy Clin immunol 108:430 (2001)).IL-17 also stimulates HBECs to release IL-6, a neutrophil-activatingfactor ( Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al.Int Arch Allergy Immunol 126:179 (2001)) and has been shown to synergizewith TNF-α to prolong the survival of human neutrophils in vitro (Laan,et al. Eur Respir J 21:387 (2003)). Moreover, IL-17 is capable ofamplifying the inflammatory responses in asthma by its ability toenhance the secretion of cytokines implicated in airway remodeling suchas the profibrotic cytokines, IL-6 and IL-11 and inflammatory mediatorsgranulocyte colony-stimulating factor (G-CSF) and granulocyte macrophagecolony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin Immunol108:430 (2001)).

Clinical evidence shows that acute, severe exacerbations of asthma areassociated with recruitment and activation of neutrophils in theairways, thus IL-17 is likely to play a significant role in asthma.Patients with mild asthma display a detectable increase in the localconcentration of free, soluble IL-17A protein (Molet, et al. J AllergyClin Immunol 108:430 (2001)) while healthy human volunteers withinduced, severe airway inflammation due to the exposure to a swineconfinement, display a pronounced increase in the concentration of free,soluble IL-17A protein in the bronchoalveolar space ( Fossiez, et al, JExp Med 183:2593 (1996), and Linden, et al. Int Arch Allergy Immunol126:179 (2001)). Furthermore, IL-17 levels in sputum have correlatedwith individuals who have increased airway hyper-reactivity Barczyk, etal. Respir Med 97:726 (2003).

In animal models of airway hyper-responsiveness, chronic inhalation ofovalbumin by sensitized mice resulted in bronchial eosinophilicinflammation and early induction of EL-17 mRNA expression in inflamedlung tissue, together with a bronchial neutrophilia Hellings, et al. AmJ Respir Cell Mol Biol 28:42 (2003). Anti-IL-17 monoclonal antibodiesstrongly reduced bronchial neutrophilic influx but significantlyenhanced IL-5 levels in both bronchoalveolar lavage fluid and serum, andaggravated allergen-induced bronchial eosinophilic influx, suggestingthat IL-17A may be involved in determining the balance betweenneutrophil and eosinophil accumulation following antigen insult Id.

Among the IL-17 family members, WL-17F is most closely related toIL-17A. The biological activities mediated by IL-17F are similar tothose of IL-17A, where IL-17F stimulates production of IL-6, IL-8 andG-CSF Hurst, et al. J Immunol 169:443 (2002). IL-17F also inducesproduction of IL-2, transforming growth factor (TGF)-□, and monocytechemoattractant protein (MCP) in endothelial cells Starnes, et al. Jrmnunol 167:4137 (2001). Similarly, allergen challenge can increaselocal IL-17F in patients with allergic asthma Kawaguchi, et al. JImmunol 167:4430 (2001). Gene delivery of IL-17F in murine lungincreases neutrophils in the bronchoalveolar space, while mucosaltransfer of the IL-17F gene enhances the levels of Ag-induced pulmonaryneutrophilia and airway responsiveness to methacholine Oda, et al. Am JRespir Crit Care Med 171:12 (2005).

Apart from asthma, several chronic inflammatory airway diseases arecharacterized by neutrophil recruitment in the airways and IL-17 hasbeen reported to play an important role in the pathogenesis ofrespiratory conditions such as chronic obstructive pulmonary disease(COPD), bacterial pneumonia and cystic fibrosis (Linden, et al. EurRespir J 15:973 (2000), Ye, et al. Am J Respir Cell Mol Biol 25:335(2001), Rahman, et al. Clin Immunol 115:268 (2005)). An anti-IL-17Aand/or anti-IL-17F therapeutic molecule could be demonstrated to beefficacious for chronic inflammatory airway disease in an in vitro modelof inflammation. The ability of antagonists to IL-17F and/or IL-17Aactivity, such as ZcytoR14 soluble receptors and antibodies theretoincluding the anti-human-ZcytoR14 monoclonal and neutralizing antibodiesof the present invention to inhibit IL-17A or and/or IL-17F-inducedcytokine and chemokine production from cultured HBECs or bronchialfibroblasts could be used as a measure of efficacy for such antagonistsin the prevention of the production of inflammatory mediators directlyresulting from IL-17A and/or F stimulation. If the addition ofantagonists to IL-17F and/or IL-17A activity, such as ZcytoR14 solublereceptors and antibodies thereto including the anti-human-ZcytoR14monoclonal and neutralizing antibodies of the present invention markedlyreduces the production and expression of inflammatory mediators, itwould be expected to be efficacious in inflammatory aspects associatedwith chronic airway inflammation.

For pharmaceutical use, the soluble ZcytoR14 or anti-ZcytoR14 antibodiesof the present invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration will be by bolus injection, controlledrelease, e.g, using mini-pumps or other appropriate technology, or byinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include a hematopoietic protein incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to provent protein loss on vial surfaces, etc.When utilizing such a combination therapy, the cytokines may be combinedin a single formulation or may be administered in separate formulations.Methods of formulation are well known in the art and are disclosed, forexample, in Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishing Co., Easton Pa., 1990, which is incorporated herein byreference. Therapeutic doses will generally be in the range of 0.1 to100 mg/kg of patient weight per day, preferably 0.5-20 mg/kg per day,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. The proteins will commonly beadministered over a period of up to 28 days following chemotherapy orbone-marrow transplant or until a platelet count of >20,000/mm³,preferably >50,000/mm³, is achieved. More commonly, the proteins will beadministered over one week or less, often over a period of one to threedays. In general, a therapeutically effective amount of soluble ZcytoR14or anti-ZcytoR14 antibodies of the present invention is an amountsufficient to produce a clinically significant increase in theproliferation and/or differentiation of lymphoid or myeloid progenitorcells, which will be manifested as an increase in circulating levels ofmature cells (e.g. platelets or neutrophils). Treatment of plateletdisorders will thus be continued until a platelet count of at least20,000/mm³, preferably 50,000/mm³, is reached. The soluble ZcytoR14 oranti-ZcytoR14 antibodies of the present invention can also beadministered in combination with other cytokines such as IL-3, -6 and-11; stem cell factor; erythropoietin; G-CSF and GM-CSF. Within regimensof combination therapy, daily doses of other cytokines will in generalbe: EPO, 150 U/kg; GM-CSF, 5-15 lg/kg; EL-3, 1-5 lg/kg; and G-CSF, 1-25lg/kg. Combination therapy with EPO, for example, is indicated in anemicpatients with low EPO levels.

Generally, the dosage of administered soluble ZcytoR14 (or ZcytoR14analog or fusion protein) or anti-ZcytoR14 antibodies will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of soluble ZcytoR14 oranti-ZcytoR14 antibodies which is in the range of from about 1 pg/kg to10 mg/kg (amount of agent/body weight of patient), although a lower orhigher dosage also may be administered as circumstances dictate.

Administration of soluble ZcytoR14 or anti-ZcytoR14 antibodies to asubject can be intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, by perfusionthrough a regional catheter, or by direct intralesional injection. Whenadministering therapeutic proteins by injection, the administration maybe by continuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising solubleZcytoR14 or anti-ZcytoR14 antibodies can be prepared and inhaled withthe aid of dry-powder dispersers, liquid aerosol generators, ornebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton etal., Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustratedby the AERX diabetes management system, which is a hand-held electronicinhaler that delivers aerosolized insulin into the lungs. Studies haveshown that proteins as large as 48,000 kDa have been delivered acrossskin at therapeutic concentrations with the aid of low-frequencyultrasound, which illustrates the feasibility of trascutaneousadministration (Mitragotri et al., Science 269:850 (1995)). Transdermaldelivery using electroporation provides another means to administer amolecule having ZcytoR14 binding activity (Potts et al., Pharm.Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising a soluble ZcytoR14 oranti-ZcytoR14 antibody can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the therapeuticproteins are combined in a mixture with a pharmaceutically acceptablecarrier. A composition is said to be a “pharmaceutically acceptablecarrier” if its administration can be tolerated by a recipient patient.Sterile phosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known to those inthe art. See, for example, Gennaro (ed.), Remington's PharmaceuticalSciences, 19th Edition (Mack Publishing Company 1995).

For purposes of therapy, soluble ZcytoR14 or anti-ZcytoR14 antibodymolecules and a pharmaceutically acceptable carrier are administered toa patient in a therapeutically effective amount. A combination of atherapeutic molecule of the present invention and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient.For example, an agent used to treat inflammation is physiologicallysignificant if its presence alleviates the inflammatory response.

A pharmaceutical composition comprising ZcytoR14 (or ZcytoR14 analog orfusion protein) or neutralizing anti-ZcytoR14 antibody can be furnishedin liquid form, in an aerosol, or in solid form. Liquid forms, areillustrated by injectable solutions and oral suspensions. Exemplarysolid forms include capsules, tablets, and controlled-release forms. Thelatter form is illustrated by miniosmQtic pumps and implants (Bremer etal., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants in DrugDelivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.), pages95-123 (CRC Press 1995); Bremer et al., “Protein Delivery with InfusionPumps,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 239-254 (Plenum Press 1997); Yewey et al., “Delivery ofProteins from a Controlled Release Injectable Implant,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117(Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPhannacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al., CancerRes. 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding ZcytoR14 activity such as ZcytoR14 monomeric,homodimeric, heterodimeric or multimeric soluble receptors, and ZcytoR14antagonists, for example anti-ZcytoR14 antibodies or bindingpolypeptides, or neutralizing anti-ZcytoR14 antibodies, which apolypeptide is linked with a polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a ZcytoR14extracellular domain, e.g., ZcytoR14 monomeric, homodimeric,heterodimeric or multimeric soluble receptors, or a ZcytoR14 antagonist(e.g., an antibody or antibody fragment that binds a ZcytoR14polypeptide, or neutralizing anti-ZcytoR14 antibody). Therapeuticpolypeptides can be provided in the form of an injectable solution forsingle or multiple doses, or as a sterile powder that will bereconstituted before injection. Alternatively, such a kit can include adry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the ZcytoR14 composition is contraindicated in patientswith known hypersensitivity to ZcytoR14.

A pharmaceutical composition comprising Anti-ZcytoR14 antibodies orbinding partners (or Anti-ZcytoR14 antibody fragments, antibody fusions,humanized antibodies and the like), or ZcytoR14 soluble receptor, can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions, aerosols, droplets, topologicalsolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Othersolid forms include creams, pastes, other topological applications, andthe like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Anti-ZcytoR14 neutralizing antibodies and binding partners with IL-17FOR IL-17A binding activity, or ZcytoR14 soluble receptor, can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly(ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modifiedAnti-ZcytoR14 antibody or binding partner, for exampleanti-Anti-ZcytoR14 antibodies or ZcytoR14 soluble receptor, linked witha polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The present invention contemplates compositions of anti-IL-17Fantibodies, and methods and therapeutic uses comprising an antibody,peptide or polypeptide described herein. Such compositions can furthercomprise a carrier. The carrier can be a conventional organic orinorganic carrier. Examples of carriers include water, buffer solution,alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.

K) Production of Transgenic Mice

Transgenic mice can be engineered to over-express the either IL-17F,IL-17A or the Zcytor14 gene in all tissues or under the control of atissue-specific or tissue-preferred regulatory element. Theseover-producers can be used to characterize the phenotype that resultsfrom over-expression, and the transgenic animals can serve as models forhuman disease caused by excess IL-17F, IL-17A or Zcytor14. Transgenicmice that over-express any of these also provide model bioreactors forproduction of Zcytor14, such as soluble Zcytor14, in the milk or bloodof larger animals. Methods for producing transgenic mice are well-knownto those of skill in the art (see, for example, Jacob, “Expression andKnockout of Interferons in Transgenic Mice,” in Overexpression andKnockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124(Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies inTransgenic Animal Science (ASM Press 1995), and Abbud and Nilson,“Recombinant Protein Expression in Transgenic Mice,” in Gene ExpressionSystems: Using Nature for the Art of Expression, Fernandez and Hoeffler(eds.), pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZcytor14 gene can begin with adult, fertile males (studs) (B6C3f1, 2-8months of age (Taconic Farms, Gerrnantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂ 5% O₂, and 90% N₂ at37° C. The egg stored in a 37° C./5% CO₂ incubator until microinjection.

Ten to twenty micrograms of plasmid DNA containing a Zcytor14 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zcytor14encoding sequences can encode a polypeptide comprising amino acidresidues 21 to 452 of SEQ ID NO:2.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zcytor14 gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zcytor14 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express IL-17F,IL-17A or Zcytor14, it is useful to engineer transgenic mice with eitherabnormally low or no expression of any of these genes. Such transgenicmice provide useful models for diseases associated with a lack ofIL-17F, IL-17A or Zcytor14. As discussed above, Zcytor14 gene expressioncan be inhibited using anti-sense genes, ribozyme genes, or externalguide sequence genes. To produce transgenic mice that under-express theZcytor14 gene, such inhibitory sequences are targeted to Zcytor14 mRNA.Methods for producing transgenic mice that have abnormally lowexpression of a particular gene are known to those in the art (see, forexample, Wu et al., “Gene Underexpression in Cultured Cells and Animalsby Antisense DNA and RNA Strategies,” in Methods in Gene Biotechnology,pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zcytor14 gene expression is to generate mice having at least onenormal Zcytor14 allele replaced by a nonfunctional Zcytor14 gene. Onemethod of designing a nonfunctional Zcytor14 gene is to insert anothergene, such as a selectable marker gene, within a nucleic acid moleculethat encodes Zcytor14. Standard methods for producing these so-called“knockout mice” are known to those skilled in the art (see, for example,Jacob, “Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Expression of the ZcytoR14 Gene

Northern analyses were performed using Human Multiple Tissue Blots(CLONTECH Laboratories, Inc., Palo Alto, Calif.). Two probes weregenerated from gel purified PCR products. The first probe was made usingZC21798 (5′ CGG CGT GGT GGT CTT GCT CTT 3′; SEQ ID NO:8) and ZC21808 (5′TCC CGT CCC CCG CCC CAG GTC 3′; SEQ ID NO:31) as primers. The probe wasa radioactively labeled using the Multiprime labeling kit from Amersham(Arlington Heights, Ill.) according to the manufacturer's protocol. Theprobe was purified using a NUCTRAP push column (STRATAGENE, La Jolla,Calif.). EXPRESSHYB (CLONTECH) solution was used for theprehybridization and hybridization solutions for the northern blots.Hybridization took place overnight at 65□C. Following hybridization, theblots were washed for 30 minutes each in solutions that contained 0.1%SDS and SSC as follows: twice in 2×SSC at room temperature, three timesin 0.1×SSC at 50 C°, once in 0.1×SSC at 55° C., and once in 0.1×SSC at65° C. The results demonstrated the ZcytoR14 gene is strongly expressedin thyroid, adrenal gland, prostate, and liver tissues, and expressed toa lesser extent in heart, small intestine, stomach, and trachea tissues.In contrast, there is little or no expression in brain, placenta, lung,skeletal muscle, kidney, pancreas, spleen, thymus, testis, ovary, colon;peripheral blood leukocytes, spinal cord, lymph node, and bone marrow.

Example 2 Distribution of mRNA in Cell Line Panels Using PCR

Total RNA was purified from resting and stimulated cell lines grownin-house and purified using a Qiagen (Valencia, Calif.) RNeasy kitaccording to the manufacturer's instructions, or an acid-phenolpurification protocol (Chomczynski and Sacchi, Analytical Biochemistry,162:156-9, 1987). The quality of the RNA was assessed by running analiquot on an Agilent Bioanalyzer. If the RNA was significantlydegraded, it was not used for subsequent creation of first strand cDNA.Presence of contam-inating genomic DNA was assessed by a PCR assay on analiquot of the RNA with zc41011 (5′CTCTCCATCCTTATCTTTCATCAAC 3′; SEQ IDNO:32) and zc41012 (5′CTCTCTGCTGGCTAAACAAAACAC 3′; SEQ ID NO:33),primers that amplify a single site of intergenic genomic DNA. The PCRconditions for the contaminating genomic DNA assay were as follows: 2.51μl 10× buffer and 0.51 μl Advantage 2 cDNA polymerase mix (BDBiosciences Clontech, Palo Alto, Calif.), 2 μl 2.5 nM dNTP mix (AppliedBiosystems, Foster City, Calif.), 2.5 μl 10× Rediload (Invitrogen,Carlsbad, Calif.), and 0.5 μl 20 uM zc41011 and zc41012, in a finalvolume of 25 ul. Cycling parameters were 94° C. 20″, 40 cycles of 94° C.20″ 60° C. 1′40 ″ and one cycle of 72° C. 7′. 10 ul od each reaction wassubjected to agarose gel electrophoresis and gels were examined forpresence of a PCR product from contaminating genomic DNA. Ifcontaminating genomic DNA was observed, the total RNA was DNAsed usingDNA-free reagents (Ambion, Inc, Austin, Tex.) according to themanufacturer's instructions, then retested as described above. Only RNAswhich appeared to be free of contaminating genomic DNA were used forsubsequent creation of first strand cDNA.

20 ug total RNA from 82 human cell lines were each brought to 98μl withH₂O, then split into two 49 ul aliquots, each containing 10 ug totalRNA, and placed in two 96-well PCR plates. To each aliquot was addedreagents for first strand cDNA synthesis (Invitrogen First Strand cDNASynthesis System, Carlsbad, Calif.): 20 μl mM MgCl₂, 10 ul 10× RTbuffer, 10 ul 0.0M DTT, 2μl oligo dT, 2ul RNAseOut. Then, to one aliquotfrom each cell line 2 μl Superscript II Reverse Transcriptase was added,and to the corresponding cell line aliquot 2 μl H₂O was added to make aminus Reverse Transcriptase negative control. All samples were incubatedas follows: 25° C. 10′, 42° C. 50′, 70° C. 15′. Samples were arranged indeep well plates and diluted to 1.7 ml with H₂O. A Multipette (Saigan)robot was used to aliquot 16.5 μl into each well of a 96-well PCR platemultiple times, generating numerous one-use PCR panels of the celllines, which were then sealed and stored at −20° C. Each well in thesepanels represents first strand cDNA from approxirnately 100 ng totalRNA. The 82 cell lines are spread across two panels, array #118A and#118B. Quality of first strand cDNA on the panels was assessed by amultiplex PCR assay on one set of the panels using primers to two widelyexpressed, but only moderately abundant genes, CLTC (clathrin) and TFRC(transferrin receptor C). 0.5 ul each of Clathrin primers zc42901(5′CTCATATTGCTCAACTGTGTGAAAAG 3′; SEQ ID NO:34),zc42902(5′TAGAAGCCACCTGAACACAAATCTG3′; SEQ ID NO:35), and TFRC primerszc42599 (5′ATCTTGCGTTGTATGTTGAAAATCAATT3′; SEQ ID NO:36), zc42600(5′TTCTCCACCAGGTAAACAAGTCTAC3′; SEQ ID NO:37), were mixed with 2.5 μl10× buffer and 0.5 μl Advantage 2 cDNA polymerase mix (BD BiosciencesClontech, Palo Alto, Calif.), 2 μl 2.5 mM dNTP mix (Applied Biosystems,Foster City, Calif.), 2.5 μl 10× Rediload (Invitrogen, Carlsbad,Calif.), and added to each well of a panel of array#118A and array#118B. Cycling parameters were as follows: 94° C. 20″, 35 cycles of 94°C. 20″, 67° C. 80″, a of 72° C. 7′. 10 μl of each reaction was subjectedto agarose gel electrophoresis and gels were scored for the presence ofa robust PCR product for each gene specific to the +RT wells for eachcell line.

Expression of mRNA in the human first strand cDNA panels for ZcytoR14was assayed by PCR with sense oligo ZC42756(5′ctctccaggcccaagtcgtgctct3′; SEQ ID NO:38) and antisense oligo ZC42757(5′ttgtcctgggggcctcgtgtctcc3′; SEQ ID NO:39) under these PCR conditionsper sample: 2.5 μl 10× buffer and 0.5 μl advantage 2 cDNA polymerase mix(BD Biosciences Clontech, Palo Alto, Calif.), 2 μl 2.5 mM dNTP mix(Applied Biosystems, ), 2.5 ul 10× Rediload (Invitrogen, Carlsbad,Calif.), and 0.5 μl 20 uM each sense and antisense primer. Cyclingconditions were 94° C. 2′, 35 cycles of 94° C. 1′, 66° C. 30″, 72° C.1.5′, and one cycle of 72° C. 7′. 10 μl of each reaction was subjectedto agarose gel electrophoresis and gels were scored for positive ornegative expression of ZcytoR14.

ZcytoR14 mRNA is widely expressed in many cell lines representing abroad spectrum of tissue and cell types. In particular, ZcytoR14 isconsistently expressed in non-T cell peripheral blood cell lines,including monocytes, B-cells, and cells of the myeloid lineage. Also,ZcytoR14 mRNA is reliably expressed in cell lines derived from skin.Other cell lines that express ZcytoR14 are all 5 of the large intestinecell lines that were present on the array.

Example 3 Distribution of mRNA in Mouse Cell Line Panels Using RT PCR

Total RNA was purified from 60 resting and stimulated cell lines grownin-house and purified using a Qiagen (Valencia, Calif.) RNeasy kitaccording to the manufacturer's instructions, an acid-phenolpurification protocol (Chomczynski and Sacchi, Analytical Biochemistry,162:156-9, 1987), or a Trizol reagent protocol (Invitrogen, Carlsbad,Calif.).

5 μg of total RNA from each cell line was arranged in a deep well96-well plate, 125 μl 3M NaOAc and 100 μl Pellet Paint (Novagen,Madison, Wis.)) were added to each well, then the final volume wasadjusted to 1.25 ml with H₂O. A Multipette (Saigan) robot was used toaliquot 25 μl of the RNA mixture followed by 75 ul EtOH into each wellof a 96-well PCR plate multiple times, generating numerous one-use RTPCR panels of the cell lines, which were then sealed and stored at −20°C. RT PCR screening was performed by first centrifuging a panel in aQiagen (Valencia, Calif.) 96-well centrifuge for 10′ at 6000 RPM.Supernatant was removed by inverting the plate onto absorbent paper. RNApellets were washed with 100 μl 70% EtOH, followed by a 5′centrifugation at 6000 RPM. Supernatant was again removed and platesallowed to air-dry until the remaining EtOH was evaporated. RNA pelletswere resuspended in 15 μl H₂O.

Expression of ZcytoR14 MRNA in the mouse cell line RNA panels wasassayed by RT PCR with zc38910 (5′acgaagcccaggtaccagaaagag3′; SEQ IDNO:40) and zc38679 (5′aaaagcgccgcagccaagagtagg3′; SEQ ID NO:41) underthese RT PCR conditions per sample: SuperScript One-Step PCR withPlatinum Taq kit, Invitrogen, Carlsbad, Calif. Cycling conditions were:1 cycle of 48° C. for 30 minutes, 94° C. for 2 minutes, followed by 35cycles of 94° C. for 15 seconds, 55° C. for 30 seconds, 72° C. for 1.5minutes, followed by 1 cycle of 72° C. for 7 minutes. 10 μl of eachreaction was subjected to agarose gel electrophoresis and gels werescored for positive or negative expression of ZcytoR14.

Murine ZcytoR14mRNA is expressed in several mouse cell lines, notably incell lines derived from bone marrow, including osteoblast, adipocyte,and preadipocyte cell lines. Also, mouse ZcytoR14 is mRNA is representedin several samples from the endocrine system, such as pancreas stromalcell lines, pancreas islet cell lines, and hypothalamus, salivary gland,and testis cell lines.

Example 4 Refolding and Purification pIL-17F Produced in E.coli

A) Inclusion Body Isolation and Extraction of pIL-17F

Following induction of protein expression in either batch ferment orshaker flask culture, the E.coli broth is centrifuged in 1 liter bottles@ 3000 RPM in a Sorvall swinging bucket rotor. Washing of the cell pasteto remove any broth contaminants is performed with 50 mM Tris pH 8.0containing 200 mM NaCl and 5 mM EDTA until the supernate is clear.

The cell pellets are then suspended in ice-cold lysis buffer (50 mM TrispH 8.0; 5 mM EDTA; 200 mM NaCl, 10% sucrose (w/v); 5 mM DTT; 5 mMBenzamidine;) to 10-20 Optical Density units at 600 nm. This slurry isthen subjected to 3 passes at 8500-9000 psi in a chilled APV 2000 LabHomogenizer producing a disrupted cell lysate. The insoluble fraction(inclusion bodies) is recovered by centrifugation of the cell lysate at20,000×G for 1 hour at 4° C.

The inclusion body pellet resulting from the 20,000×G spin is weighedand then re-suspended in wash buffer (50 mM Tris pH 8 containing 200 mMNaCl, 5 mM EDTA, 5 mM DTT, 5 mM Benzamidine ) at 10 ml wash buffer pergram inclusion bodies. Complete dispersion is achieved by homogenizingwith an OMNI international rotor stator generator. This suspension iscentrifuged at 20,000×G for 30 minutes at 4° C. The wash cycle isrepeated 3-5 times until the supernatant is clear.

The final washed pellet is solubilized in 7M Guanidine HCl in 40 mM Trisbuffer at pH 8 containing 0.1M Sodium Sulfite and 0.02 M SodiumTetrathionate. The extraction and sulfitolysis reaction is allowed toproceed with gentle stirring at 4° C. overnight. The resulting pinkishcolored solution is centrifuged at 35,000×g for 1 hour at 4° C. and theclarified supernate, containing the soluble pIL-17F, is 0.45 umfiltered.

B) pIL-17F Refolding Procedure

The solubilized, sulfitolyzed pIL-17F is refolded by drop wise dilutioninto ice cold refolding buffer containing 55 mM MES, 10.56 mM NaCl, 0.44mM KCl, 0.055% PEG (3400 K), 1.1 mM EDTA, 20% Glycerol, 0.5M GuanidineHCl, 0.75 M Arginine and the Glutathione redox pair at a 1:1 ratio (1 mMGSH: 1 mM GSSG ). The pH of the refolding buffer is adjusted to 6.5 withHCl and the pIL-17F is added to a final concentration of 100 ug/ml. Oncediluted, the mixture is allowed to stir slowly in the cold room for 72hours.

C) Product Recovery & Purification

The refolded pIL-17F is concentrated 10× vs. a 10 kDa cutoff membrane ona lab scale TFF system. Next it is filtered using a 0.45 micron membraneand the pH is adjusted to 5.1 with the addition of Acetic acid. ThepH-adjusted material is captured by cation exchange chromatography on aPharmacia SP Fast Flow column equilibrated in 50 mM Acetate buffer, pH5.1. The pIL-17F is loaded by inline proportioning at 1:5 withequilibration buffer at a flow rate of 190 cm/hr. This dilution lowersthe ionic strength enabling efficient binding of the target to thematrix. After sample loading is complete, the column is washed tobaseline absorbance with equilibration buffer. The column is washed with0.4 M NaCl in 50 mM Acetate buffer at pH 5.1 and then the bound proteinis eluted with a 5 CV gradient from 0.4 M to 1.5 M NaCl in 50 mM Acetatebuffer at pH 5.1. The protein elutes at ˜1M NaCl and is approximately85% dimeric by SDS PAGE analysis of eluate fractions. The fractionscontaining pIL-17F are pooled and concentrated against a 10 kDa cutoffultrafiltration membrane using an Amicon stirred cell in preparation forthe final purification and buffer exchange by size exclusionchromatography.

D) Size Exclusion Buffer Exchange and Formulation

The concentrated cation pool (at a volume of 3-4% of CV) is injected ata flow rate of 30 cm/hr onto a Pharmacia Superdex 75 size exclusioncolumn equilibrated in 50 mM Sodium Phosphate buffer containing 109 mMNaCl, pH 7.2. The symmetric eluate peak containing the product isdiluted to a concentration of 1 mg/ml in 50 mM Sodium Phosphate buffercontaining 109 mM NaCl, pH 7.2. Finally the pIL-17F is 0.2 micronsterile filtered, aliquoted and stored at ˜80° C. The final processyield is 20%.

Example 5 Construction of Mammalian Soluble ZcytoR14 ExpressionConstruct

An expression construct containing human ZcytoR14 [L21-K451]-mFc1 (mouseBALB/c μ2a Fc) is constructed via overlap PCR and homologousrecombination using a DNA fragment (SEQ ID NO:42) encoding a ZcytoR14polypeptide (SEQ ID NO:43), a DNA fragment encoding mFc1 (SEQ ID NO:44),and the expression vector pZMP20. The fragments are generated by PCRamplification.

The PCR fragment encoding ZcytoR14 [L21-K45 1] contains a 5′ overlapwith the pZMP20 vector sequence in the optimized tissue plasminogenactivator pre-pro secretion leader sequence coding region, the ZcytoR14extracellular domain coding [L21-K451], and a 3′ overlap with the mFclcoding region. The PCR amplification reaction uses the 5′oligonucleotide [GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGAGGCTTGTGGGGCCT; SEQ ID NO:46], the 3′ oligonucleotide[TGTGGGCCCTCTGGGCTCCTTGTGGATGTATTTGTC; SEQ ID NO:47], and a previouslygenerated DNA clone of ZcytoR14 as the template.

The PCR fragment encoding mFcl contains a 5′ overlap with the ZcytoR14sequence, the mFc1 coding region, and a 3′ overlap with the pZMP20vector in the poliovirus internal ribosome entry site region. The PCRamplification reaction uses the 5 oligonucleotide[GACAAATACATCCACAAGGAGCCCAGAGGGCCCACA; SEQ ID NO:48], the 3′oligonucleotide[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTTACCCGGAGTCCGGGA; SEQ ID NO:49],and a previously generated DNA clone of mFc1 as the template.

The PCR amplification reaction conditions are as follows: 1 cycle, 94°C., 5 minutes; 35 cycles, 94° C., 1 minute, followed by 55° C., 2minutes, followed by 72° C., 3 minutes; 1 cycle, 72° C., 10 minutes. ThePCR reaction mixtures are run on a 1% agarose gel and the DNA fragmentscorresponding to the expected sizes are extracted from the gel using aQlAquickTm Gel Extraction Kit (Qiagen, Cat. No. 28704).

The two PCR fragments are joined by overlap PCR. Approximately 1 μl eachof the two gel extracted fragments are combined in a PCR amplificationreaction using the 5′ oligonucleotide[GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGA GGCTTGTGGGGCCT;SEQ ID NO:46] and the 3′ oligonucleotide[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTTACCCGGAGTCCGGGA; SEQ ID NO:49).PCR conditions used are as follows: 1 cycle, 94° C., 5 minutes; 35cycles, 94° C., 1 minute, followed by 55° C., 2 minutes, followed by 72°C., 3 minutes; 1 cycle, 72° C., 10 minutes. The PCR reaction mixture isrun on a 1% agarose gel and the DNA fragment corresponding to the sizeof the insert is extracted from the gel using a QIAquick™ Gel ExtractionKit (Qiagen, Cat. No. 28704).

Plasmid pZMP20 is a mammalian expression vector containing an expressioncassette having the MPSV promoter, a BglII site for linearization priorto yeast recombination, an otPA signal peptide sequence, an internalribosome entry element from poliovirus, the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain; an E. coliorigin of replication; a mammalian selectable marker expression unitcomprising an SV40 promoter, enhancer and origin of replication, a DHFRgene, and the SV40 terminator; and URA3 and CEN-ARS sequences requiredfor selection and replication in S. cerevisiae.

The plasmid pZMP20 is digested with BglII prior to recombination inyeast with the gel extracted ZcytoR14[L21-K451]-mFc1 PCR fragment. 100μl of competent yeast (S. cerevisiae) cells are combined with 10 μl ofthe ZcytoR14[L21-K451]-mFc1 insert DNA and 100 ng of BglII digestedpZMP20 vector, and the mix is transferred to a 0.2 cm electroporationcuvette. The yeast/DNA mixture is electropulsed using power supply(BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∝ohms, and 25 μF. Six hundred til of 1.2 M sorbitol is added to thecuvette, and the yeast is plated in 100 μl and 300 μl aliquots onto twoURA-D plates and incubated at 30° C. After about 72 hours, the Ura⁺yeast transformants from a single plate are resuspended in 1 ml H₂O andspun briefly to pellet the yeast cells. The cell pellet is resuspendedin 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). The five hundred μl of the lysis mixture isadded to an Eppendorf tube containing 250 μl acid-washed glass beads and300 μl phenol-chloroform, is vortexed for 3 minutes, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundred μl ofthe aqueous phase is transferred to a fresh tube, and the DNA isprecipitated with 600 μl ethanol, followed by centrifugation for 30minutes at maximum speed. The tube is decanted and the pellet is washedwith 1 mL of 70% ethanol. The tube is decanted and the DNA pellet isresuspended in 30 μl 10 mM Tris, pH 8.0, 1 mM EDTA.

Transformation of electrocompetent E. coli host cells (DH12S) is doneusing 5 μl of the yeast DNA preparation and 50 μl of E. coli cells. Thecells are electropulsed at 2.0 kV, 25 μF, and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) is added and then the cells are plated in 50 μland 200 μl aliquots on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™Agar (Difco), 100 mg/L Ampicillin).

The inserts of three DNA clones for the construct is subjected tosequence analysis and one clone containing the correct sequence isselected. Large scale plasmid DNA is isolated using a commerciallyavailable kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia, Calif.)according to manufacturer's instructions.

Example 6 Construction of Mammalian Soluble ZcytoR14 ExpressionConstructs that Express ZcytoR14-CEE, ZcytoR14-CHIS, and ZcytoR14-CFLAG

An expression construct containing human ZcytoR14 [L21-K451] with aC-terminal tag, either Glu-Glu (CEE), six His (CHIS), or FLAG (CFLAG),is constructed via PCR and homologous recombination using a DNA fragmentencoding ZcytoR14 [L21-K451] (SEQ ID NO:42) and the expression vectorpZMP20.

The PCR fragment encoding ZcytoR14CEE contains a 5′ overlap with thepZMP20 vector sequence in the optimized tissue plasminogen activatorpre-pro secretion leader sequence coding region, the ZcytoR14extracellular domain coding [L21-K451], the sequence of the Glu-Glu tag(Glu Glu Tyr Met Pro Met Glu; SEQ ID NO:53), and a 3′ overlap with thepZMP20 vector in the poliovirus internal ribosome entry site region. ThePCR amplification reaction uses the 5′ oligonucleotide[GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGA GGCTTGTGGGGCCT;SEQ ID NO:46], the 3′ oligonucleotide[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTCCATGGGCATGTATTCTTCCTTGTGGATGTATTTGTC; SEQ ID NO:50], and a previously generated DNAclone of ZcytoR14 as the template.

The PCR amplification reaction condition is as follows: 1 cycle, 94° C.,5 minutes; 35 cycles, 94° C., 1 minute, followed by 55° C., 2 minutes,followed by 72° C., 3 minutes; 1 cycle, 72° C., 10 minutes. The PCRreaction mixture is run on a 1% agarose gel and the DNA fragmentcorresponding to the expected size is extracted from the gel using aQIAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704).

The plasmid pZMP20 is digested with BglII prior to recombination inyeast with the gel extracted ZcytoR14CEE PCR fragment. One hundred μl ofcompetent yeast (S. cerevisiae) cells are combined with 10 μl of theZcytoR14CEE insert DNA and 100 ng of BglII digested pZMP20 vector, andthe mix is transferred to a 0.2 cm electroporation cuvette. Theyeast/DNA mixture is electropulsed using power supply (BioRadLaboratories, Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∝ ohms,and 25 μF. Six hundred μl of 1.2 M sorbitol is added to the cuvette, andthe yeast is plated in 100 μl and 300 μl aliquots onto two URA-D platesand incubated at 30° C. After about 72 hours, the Ura⁺ yeasttransformants from a single plate are resuspended in 1 ml H₂O and spunbriefly to pellet the yeast cells. The cell pellet is resuspended in 0.5ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH8.0, 1 mM EDTA). The five hundred il of the lysis mixture is added to anEppendorf tube containing 250 μl acid-washed glass beads and 300 μlphenol-chloroform, is vortexed for 3 minutes, and spun for 5 minutes inan Eppendorf centrifuge at maximum speed. Three hundred μl of theaqueous phase is transferred to a fresh tube, and the DNA isprecipitated with 600 μl ethanol, followed by centrifugation for 30minutes at maximum speed. The tube is decanted and the pellet is washedwith 1 mL of 70% ethanol. The tube is decanted and the DNA pellet isresuspended in 30 μl 10 mM Tris, pH 8.0, 1 mM EDTA.

Transformation of electrocompetent E. coli host cells (DH12S) is doneusing 5 μl of the yeast DNA preparation and 50 μl of E. coli cells. Thecells are electropulsed at 2.0 kV, 25 μF, and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) is added and then the cells are plated in 50 μland 200 μl aliquots on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™Agar (Difco), 100 mg/L Ampicillin).

The inserts of three DNA clones for the construct is subjected tosequence analysis and one clone containing the correct sequence isselected. Large scale plasmid DNA is isolated using a commerciallyavailable kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia, Calif.)according to manufacturer's instructions.

The same process is used to prepare the ZcytoR14 with a C-terminal histag, composed of Gly Ser Gly Gly His His His His His His (ZcytoR14CHIS;SEQ ID NO:5 1) or the C-terminal FLAG tag, composed of Gly Ser Asp TyrLys Asp Asp Asp Asp Lys (ZcytoR14CFLAG; SEQ ID NO:52). To prepare theseconstructs, instead of the 3′ oligonucleotide of SEQ ID NO:50; the 3′oligonucleotide [CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTAGTGATGGTGATGGTGATGTCCACCAGATCCCTTGTGGATGTATTTGTC; SEQ ID NO:54] is used to generateZcytoR14CHlS or the 3′ oligonucleotide[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTACTTATCATCATCATCCTTATAATCGGATCCCTTGTGGATGTATTTGTC; SEQ ID NO:55] is used to generateZcytoR14CFLAG.

Example 7 Transfection and Expression of Soluble ZcytoR14 ReceptorExpression Constructs that Express the ZcytoR14-mFc1 Fusion Protein, andthe ZcytoR14-CEE, ZcytoR14-CHIS, and ZcytoR14-CFLAG C-Terminal TaggedProteins

Three sets of 200 μg of each of the soluble ZcytoR14 fusion or taggedexpression constructs are separately digested with 200 units of PvuI at37° C. for three hours, precipitated with isopropyl alcohol, andcentrifuged in a 1.5 mL microfuge tube. The supernatant is decanted offthe pellet, and the pellet is washed with 1 mL of 70% ethanol andallowed to incubate for 5 minutes at room temperature. The tube is spunin a microfuge for 10 minutes at 14,000 RPM and the supernatant isdecanted off the pellet. The pellet is then resuspended in 750 μl of CHOcell tissue culture medium in a sterile environment, allowed to incubateat 60° C. for 30 minutes, and is allowed to cool to room temperature.Approximately 5×10⁶ CHO cells are pelleted in each of three tubes andare resuspended using the DNA-medium solution. The DNA/cell mixtures areplaced in a 0.4 cm gap cuvette and electroporated using the followingparameters; 950 μg, high capacitance, at 300 V. The contents of thecuvettes are then removed, pooled, and diluted to 25 mLs with CHO celltissue culture medium and placed in a 125 mL shake flask. The flask isplaced in an incubator on a shaker at 37° C., 6% CO₂ with shaking at 120RPM.

The CHO cells are subjected to nutrient selection followed by stepamplification to 200 nM methotrexate (MTX), and then to 1 μM MTX. Fusionor tagged protein expression is confirmed by Western blot, and the CHOcell pool is scaled-up for harvests for protein purification.

Example 8 Expression of Soluble ZcytoR14

An expression plasmid containing ZcytoR14-Tbx-C(Fc9) (SEQ ID NO:64) wasconstructed via homologous recombination using a DNA fragment ofZcytoR14 _Tbx and the expression vector pZMP40. The fragment wasgenerated by PCR amplification using primers zc44531 and zc44545.

The PCR fragment ZcytoR14_Tbx contains a partial ZcytoR14 extracellulardomain coding region, which was made using a previously generated cloneof ZcytoR14 as the template. The fragment includes a 5′ overlap with thepZMP40 vector sequence in the otPA coding region, the ZcytoR14 segment(amino acid residue 21 to 451 of SEQ ID NO:2), a linker sequence, athrombin cleavage site, and a 3′ overlap with the pZMP40 vector in theFc9 coding region. PCR conditions used were as follows: 1 cycle, 94° C.,5 minutes; 35 cycles, 94° C., 1 minute, followed by 55° C., 2 minutes,followed by 72° C., 3 minutes; 1 cycle, 72° C., 10 minutes.

The PCR reaction mixtures were run on a 1% agarose gel and a bandcorresponding to the sizes of the inserts were gel-extracted using aQlAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704).

Plasmid pZMP40 is a mammalian expression vector containing an expressioncassette having the MPSV promoter, multiple restriction sites forinsertion of coding sequences, an otPA signal peptide sequence, and thesequence for Fc9; an internal ribosome entry site (IRES) element frompoliovirus, and the extracellular domain of CD8 truncated at theC-terminal end of the transmembrane domain; an E. coli origin ofreplication; a mammalian selectable marker expression unit comprising anSV40 promoter, enhancer and origin of replication, a DHFR gene, and theSV40 terminator; and URA3 and CEN-ARS sequences required for selectionand replication in S. cerevisiae. It was constructed from pZMP21 (PatentPub. No. US 2003/0232414 A1; deposited at the American Type CultureCollection and designated as ATCC# PTA-5266).

The plasmid pZMP40 was cut with BglIl prior to recombination in yeastwith the PCR fragment. One hundred microliters of competent yeast (S.cerevisiae) cells were independently combined with 10 μl of the insertDNA (SEQ ID NO:66) and 100 ng of cut pZMP40 vector, and the mix wastransferred to a 0.2-cm electroporation cuvette. The yeast/DNA mixturewas electropulsed using power supply (BioRad Laboratories, Hercules,Calif.) settings of 0.75 kV (5 kV/cm), ∝ ohms, and 25 μF. Six hundred μlof 1.2 M sorbitol was added to the cuvette, and the yeast was plated ina 100-μl and ³⁰⁰ μl aliquot onto two URA-D plates and incubated at 30°C. After about 72 hours, the Ura⁺ yeast transformants from a singleplate were resuspended in 1 ml H₂O and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 0.5 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Thefive hundred microliters of the lysis mixture was added to an Eppendorftube containing 250 A¹ acid-washed glass beads and 300 μlphenol-chloroform, was vortexed for 3 minutes, and spun for 5 minutes inan Eppendorf centrifuge at maximum speed. Three hundred microliters ofthe aqueous phase was transferred to a fresh tube, and the DNA wasprecipitated with 600 μl ethanol (EtOH), followed by centrifugation for30 minutes at maximum speed. The tube was decanted and the pellet waswashed with 1 mL of 70% ethanol. The tube was decanted and the DNApellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (DH12S) was doneusing 5 μl of the yeast DNA prep and 50 I¹I of cells. The cells wereelectropulsed at 2.0 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mnM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and a 200μl aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of three clones for the construct was subjected to sequenceanalysis and one clone for each construct, containing the correctsequence, was selected. Larger scale plasmid DNA was isolated using acommercially available kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia,Calif.) according to manufacturer's instructions.

Three sets of 200 μg of the ZcytoR14[L21-K451]_Tbx-C(Fc9) construct werethen each digested with 200 units of Pvu I at 37° C. for three hours andthen were precipitated with IPA and spun down in a 1.5 mL microfugetube. The supernatant was decanted off the pellet, and the pellet waswashed with 1 mnL of 70% ethanol and allowed to incubate for 5 minutesat room temperature. The tube was spun in a microfuge for 10 minutes at14,000 RPM and the supernatant was decanted off the pellet. The pelletwas then resuspended in 750 μl of PF-CHO media in a sterile environment,allowed to incubate at 60° C. for 30 minutes, and was allowed to cool toroom temperature. 5E6 APFDXB11 cells were spun down in each of threetubes and were resuspended using the DNA-media solution. The DNA/cellmixtures were placed in a 0.4 cm gap cuvette and electroporated usingthe following parameters: 950 μF, high capacitance, and 300 V. Thecontents of the cuvettes were then removed, pooled, and diluted to 25mLs with PF-CHO media and placed in a 125 mL shake flask. The flask wasplaced in an incubator on a shaker at 37° C., 6% CO₂, and shaking at 120RPM.

The cell line was subjected to nutrient selection followed by stepamplification to 200 nM methotrexate (MTX), and then to 1 μM MTX.Expression was confirmed by western blot, and the cell line wasscaled-up and protein purification followed.

Example 9 Purification of Soluble ZcytoR14 from CHO Cells

Conditioned media from CHO cells expressing ZcytoR14-TbX-Fc9 (SEQ IDNO:64) was concentrated approximately 10-fold with a Pellicon-IItangential flow system against two Biomax 0.1 m² 30 kD molecular weightcutoff membrane cassettes (Millipore, Bedford, Mass.). The concentratedmedia was pH adjusted to 5.5 with glacial acetic acid, 0.2 □m sterilefiltered then loaded onto a Protein G sepharose fast flow resin(Pharmacia, Piscataway, N.J.) via batch chromatography overnight at 4 C.Prior to loading the pH adjusted conditioned media, the Protein G resinwas pre-equilibrated with, 5 column volumes (approximately 150 ml) of 25mM sodium acetate, 150 mM NaCl, pH5.5. The ratio of filtered, pHadjusted conditioned media to resin was 33:1 (v/v).

The batched chromatography process was performed at ambient roomtemperature (approximately 21 C). The batched, pH adjusted, 0.22 μmfiltered, conditioned media was poured into an empty 5.5×20.5 cm glasscolumn (BioRad, Hercules, Calif.) and packed via gravity. The column waswashed with 10 column volumes (approximately 300 ml) of 25 mM sodiumacetate, 150 mM NaCl, pH5.5. Bound protein was then pH eluted with lOOmMglycine, pH 2.7. 9.0 ml fractions were collected and immediatelyneutralized with 1.0 ml 2.0M Tris, pH 8.0. The collected fractions wereanalyzed via SDS-PAGE Coomassie staining. Fractions containingZcytoR14-Tbx-Fc9 were pooled and concentrated approximately 6-fold usinga 5 kD molecular weight cutoff Biomax membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

The pooled, concentrated fractions were then dialyzed, at 4 C,extensively against 1× phosphate buffered saline, pH 7.3 (Sigma, St.Louis, Mo.) using a 7 kD molecular weight cutoff membrane Slide-A-Lyzer(Pierce, Rockford, Ill.). ZcytoR14-TbX-Fc9 as formulated in 1× phosphatebuffered saline, pH 7.3 was 0.22 μm sterile filtered prior to aliquotingand storage at −80 C.

Example 10 Binding of IL-17A and IL-17F to Human ZcytoR14

A) Binding of Biotinylated Cytokines to Transfected Cells

Baby Hamster Kidney (BHK) cells that had been transfected withexpression vectors encoding human IL-17 receptor (SEQ ID NO:21), humanZcytoR14 (SEQ ID NO:2), or both of these receptors are assessed fortheir ability to bind biotinylated human IL-17A and human IL-17F. Cellsare harvested with versene, counted and diluted to 10⁷ cells per ml instaining media (SM), which is HBSS plus 1 mg/ml bovine serum albumin(BSA), 10 mM Hepes, and 0.1% sodium azide (w/v). Biotinylated humanIL-17A (SEQ ID NO:14) and human IL-17F (SEQ ID NO:16) are incubated withthe cells on ice for 30 minutes at various concentrations. After 30minutes, excess cytokine is washed away with SM and the cells areincubated with a 1:100 dilution of streptavidin conjugated tophycoerythrin (SA-PE) for 30 minutes on ice. Excess SA-PE is washed awayand cells are analyzed by flow cytometry. The amount of cytokine bindingwas quantitated from the mean fluorescence intensity of the cytokinestaining. From this analysis, we find that human IL-17A binds both thehuman IL-17R and ZcytoR14 to a similar extent. Also, human IL-17F bindsZcytoR14 to a similar level, but binds IL-17R detectably, but to a muchlower level than was seen with IL-17A.

B) Binding of Biotinylated Cytokines to Human Peripheral BloodMononuclear Cells

Human peripheral blood mononuclear cells (PBMC) were prepared from wholeblood by ficoll density gradient centrifugation. PBMC at 10⁷ cells perml were simultaneously incubated with biotinylated IL-17A or IL-17F at 1μg/ml and fluorochrome conjugated antibodies to specific cell surfaceproteins that were designed to distinguish various white blood celllineages lineages. These markers include CD4, CD8, CD19, CD11b, CD56 andCD16. Excess antibody and cytokine are washed away, and specificcytokine binding is detected by incubating with SA-PE as describedabove. Samples were analyzed by flow cytometry and from this analysis,we find that human IL-17A binds to virtually all PBMC populationsexamined, but that human IL-17F does not detectably bind to anypopulation.

C) Inhibition of Specific Binding of Biotinlyated Human IL-17A andEL-17F with Unlabeled Cytokine

Binding studies are performed as discussed above, but excess unlabeledhuman IL-17A and IL-17F are included in the binding reaction. In studieswith BHK cells, the amount of unlabeled cytokine was varied over a rangeof concentrations and we find that addition of unlabeled IL-17A competedfor binding of both IL-17A and IL-17F to both ZcytoR14 and IL-17R.However, unlabeled IL-17F competed for binding of both IL-17A and IL-17Fto ZcytoR14, but it did not compete effectively for binding to IL-17R.This indicates that both IL-17A and IL-17F specifically bind toZcytoR14, and that they bind at a site that is either identical oroverlaps significantly since they cross-compete for binding. Also,IL-17A competes for the relatively weak binding of IL-17F for IL-17R,indicating these two cytokines also bind to a similar region in theIL-17R, but IL-17F binds IL-17R with much reduced affinity relative toZcytoR14.

D) Inhibition of Specific Binding of Biotinylated Human IL-17A andIL-17F with Soluble ZcytoR14 and IL-17R

Binding studies are performed as discussed above, except that a solubleform of ZcytoR14 or IL-17R are included in the binding reactions. Thesesoluble receptors are fusion proteins derived from the extracellulardomain of each receptor fused to the human IgG1 constant (Fc) region. Wefind that soluble ZcytoR14 inhibits binding of both human IL-17A andIL-17F to both IL-17R and ZcytoR14 transfected BHK cells. However,soluble IL-17R inhibits binding of IL-17A to either receptor, but doesnot effectively block binding of IL-17F to ZcytoR14, consistent with thepoor binding of IL-17F for the IL-17R.

Example 11 IL-17A and IL-17F Bind to ZcytoR14

A) Binding Inhibition with Cold Ligand

BHK cells transfected with hZcytoR14 (SEQ ID NO:2) and IL-17R (SEQ IDNO:21) were plated at 40,000 cells/well in a 24-well dish (Costar3527)two days prior to assay. IL-17A (SEQ ID NO:14) and IL-17F(SEQ IDNO:16) that had been radiolabeled by the iodobead method were addedindependently to wells in triplicate at 10 ng/ml with a total of 250ul/well in binding buffer (RPMI 1640 media (JRH 51502-500M) with 10mg/ml bovine serum albumin(Gibcol5260-037)). Cold competitors were addedin 100 fold molar excess. Competitors tested included IL-17A, IL-17B,IL-17C, IL-17D, IL-17E, IL-17F and IL-21. Wells were incubated on icefor 1-hour followed by two washes with PBS (Invitrogen 20012-027) andone wash with a high salt solution (1.5M NaCL, 50 mM HEPES pH 7.4).Wells were extracted with 500 ul of 0.8M NaOH for 30 min. at roomtemperature and counts per minute were measured in a gamma counter(Packard Cobra II A5005).

The results indicated that 100x molar cold I-17A and IL-17F were able toreduce binding of ¹²⁵I IL-17A to BHK hZcytoR14 by approximately 7 foldwhile IL-17B,C,D,E and IL-21 had no effect on binding. 100x molar coldIL-17A reduced the binding of ¹²⁵IL-17A to BHK IL-17R by approximately 4fold while IL-17B,C,D,E,F and IL-21 had no effect on binding. 100x molarcold IL-17A and IL-17F reduced the binding of ¹²⁵IL-17F to BHK hZcytoR14by approximately 4 fold and 5 fold, respectively, while IL-17B,C,D,E andIL-21 had no effect on binding.

B) Binding Inhibition with Soluble Recepto:

Binding to hzytor14 (SEQ ID NO:2) and IL-17R (SEQ ID NO:21) transfectedBHK cells was performed as in one, but 100 fold molar excess solublehZcytoR14x1/Fc9 (Example 8) and soluble IL-17R/Fc (obtained from R&D;Ref. 177-IR) were used in place of cold ligand in the competition. Cellswere washed, extracted and counted as in part one.

Soluble hZcytoR14/F_(C) inhibited binding of 1²⁵IL-17F to BHK hZcytoR14with an IC₅₀ of 10× molar excess average from three experiments. SolublehZcytoR14/F_(C) inhibition of ¹²⁵IIL-17A on the same cell line gave anaverage IC₅₀ of 20× molar excess and soluble IL-17R/Fc inhibition of125I IL-17A gave an average IC₅₀ of 20× molar excess.

C) Binding Saturation

Transfected BHK cells were plated into 24-well dishes as in one.Radiolabeled IL-17A and IL-17F were added starting at a concentration of4 nM in eight 1:3 dilutions (to a concentration of 1.83 pM) intriplicate with a total of 2501 μl/well in binding buffer. Separately,100 fold molar excess of cold ligand was added at each dilution point.Cells were washed, extracted and counted as in one. Specific counts perminute were plotted against concentration of radiolabeled ligand addedby subtracting the 100 fold excess counts from the the uncompeted countsat each dilution point. These normalized data were plotted to generatesaturation binding curves for each combination of radiolabeled ligandand transfected BHK cells. Table 4 shows the affinity values calculatedfrom all three experiments. TABLE 4 125I IL-17A + BHK hZcytoR14 125IIL-17A + BHK IL-17R 1. 180 pM 1. 2.5 +/− 0.2 nM 2. 200 pM 2. 4.5 +/− 0.3nM 3. 370 pM 3. 5.9 +/− 0.1 nM 125I IL-17F + BHK hZcytoR14 125I IL-17F +BHK IL-17R 1. 50 pM 1. Very low affinity 2. 60 pM 2. Very low affinity3. 80 pM 3. Very low affinityOne-site binding curve fits agreed most closely with IL-17A & IL-17Fbinding to IL-17R. Two-site binding curve fits agreed most closely withIL-17A and IL-17F binding to hZcytoR14. The high affinity binding siteis the value shown above. The low affinity binding site had very lowaffinity and varied widely between the three experiments.

Example 12 Murine Nih3t3 Cells Respond to Human IL-17A and IL-17F

A) Cell Plating and kz142 Adenovirus Reporter Infection.

Nih3t3 cells, derived from mouse fibroblasts (described in ATCC) Nih3t3were plated at 5000 cells/well in solid white, cell culture coated 96well plates, (Cat. #3917. Costar) using DMEM/10% FBS, containingglutamine and amended with pyruvate and cultured overnight at 37oC and5% C02. On this second day, the plating media was removed and Kz142adenovirus particles at a multiplicity of infection of 5000particles/cell were prepared in DMEM/1% FBS, containing glutamine andamended with pyruvate and cultured overnight at 37oC and 5% C02.

B) Luciferase Assay Measuring IL-17A and F Activation of kz142Adenovirus Reporter Infected nih3t3 Cells.

Following the overnight incubation with the adenovirus particlereporter, human IL-17A and IL-17F Ligand treatments were prepared inserum free media ( )amended to 0.28% BSA. The adenovirus particles andmedia were removed and the appropriate ligand doses were given intriplicates. Incubation at 37° C. and 5% C02 was continued for 4 hours,after which the media was removed, cells lysed for 15 minutes and meanfluorescence intensity (MFI) measured using the luciferase assay systemand reagents. (Cat.#e1531 Promega. Madison, Wis.) and a Microplateluminometer. Activity was detected at concentrations ranging from0.1-1000 ng/ml human IL-17A and IL-17F, generating EC50 values of about50 ng/ml for both ligands. These data suggest that nih3t3 cells carryreceptors to these ligands and that IL-17A and IL-17F activate theNfkb/Ap-1 transcription factor.

Example 13 Murine Nih3t3 Cells Express Both IL-17 Receptor and ZcytoR14Receptor

RTPCR analysis of nih3t3 RNA demonstrated that these cells are positivefor both IL-17 Receptor and ZcytoR14 receptor, consistent with theirnfkb/ap1 response to human IL-17A and IL-17F mediation being mediatedthrough one or both of these receptors.

RTPCR DETAILS:

A) Mouse cytor14 PCR

First strand cDNA was prepared from total RNA isolated from nih3t3 cellsusing standard methods. PCR was applied using hot star polymerase andthe manufacturer's recommendations (Qiagen, Valencia, Calif.) usingsense primer, zc38910, 5′ ACGAAGCCCAGGTACCAGAAAGAG 3′ (SEQ ID NO:56) andantisense primer, zc 38679, 5′ AAAAGCGCCGCAGCCAAGAGTAGG 3′ (SEQ IDNO:57) and 35 cycles of amplification. Agarose gel electrophoresisrevealed a single, robust amplicon of the expected, 850 bp size.

B) Mouse IL-17R PCR

First strand cDNA was prepared from total RNA isolated from nih3t3 cellsusing standard methods. PCR was applied using hot star polymerase andthe manufacturer's recommendations (Qiagen, Valencia, Calif.) usingsense primer, zc38520, 5′ CGTAAGCGGTGGCGGTTTTC 3′(SEQ ID NO:58) andantisense primer, zc 38521, 5′ TGGGCAGGGCACAGTCACAG 3′ (SEQ ID NO:59)and 35 cycles of amplification. Agarose gel electrophoresis revealed asingle, robust amplicon of the expected, 498 bp size.

Example 14 Creation of a Stable Nih3t3 Assay Clone Expressing theapl/nfkb Transcription Factor

The murine nih3t3 cell line described above was stably transfected withthe kz142 ap1/nfkb reporter construct, containing a neomycin-selectiblemarker. The Neo resistant transfection pool was plated at clonaldensity. Clones were isolated using cloning rings and screened byluciferase assay using the human IL-17A ligand as an inducer. Cloneswith the highest mean fluorescence intensity (MFI) (via ap1/NfkBluciferase) and the lowest background were selected. A stabletransfectant cell line was selected and called nih3t3/kz142.8.

Example 15 Inhibition of Activation by Human IL-17A and IL-17F in MurineNih3t3 Cells Using Soluble ZcytoR14 and IL-17 Receptor/FC Chimeras

Soluble forms of ZcytoR14 or IL-17R were used as antagonists of humanIL-17A and IL-17F activation of ap1/nfkb elements in a luciferase assay.These soluble receptors are fusion proteins derived from theextracellular domain of each receptor fused to the human IgGl constant(Fc) region. The soluble human IL-17R FC fusion protein was purchased.(recombinant human IL-17R/FC chimera, catalog number 177-IR-100, R&DSystems, Inc., Minneapolis, Minn.) The soluble human ZcytoR14 FC chimera(ZcytoR14sR/FC9) was constructed as described above. We find that anexcess ZcytoR14sR/FC9 and human IL17RsR/FC chimera inhibit EC50 levelsof both human IL-17A and IL-17F mediation of apl/nfkb activation of themurine nih3t3/kz142.8 assay cell line.

The ZcytoR14sR/FC9 protein showed the greatest potency in antagonizingIL-17F activation and IL17RsR/FC chimera showed the greatest potency inantagonizing IL-17A activation.

Example 16 IL-17F mRNA is Upregulated in a Murine Model of Asthma

IL-17F mRNA levels were measured in a sensitization and airway challengemodel in mice. Groups of mice, 8 to 10 wks of age, were sensitized byintraperitoneal injection of 10 ug of recombinant Dernatophagoidespteronyssinus allergen 1 (DerP1) (Indoor biotechnologies, Cardiff, UK)in 50 % Imject Alum (Pierce) on days 0 and 7. Seven days later, micewere challenged on 3 consecutive days (days 14, 15 and 16) with 20 ug ofDerP1 in 50 ul PBS. There were 4 mice representing this group. Negativecontrols included 5 mice given phosphate buffered saline (PBS)sensitization, followed by PBS challenge. In addition to 3 mice givenDerP1 sensitization, followed by PBS challenge. Forty-eight hoursfollowing allergen, or control challenge whole lung tissue was harvestedand total RNA was isolated.

First strand cDNA was prepared using identical amounts of total RNA fromeach subject. IL-17F PCR was applied using Qiagen hotstar polymerase(Qiagen, Valencia, Calif.) and the manufacturer's recommendations. TheIL-17F PCR utilized 35 cycles of amplification with sense primer,zc46098, 5′ ACTTGCCATTCTGAGGGAGGTAGC 3′ (SEQ ID NO:60) and antisenseprimer, 46099, 5′ CACAGGTGCAGCCAACTTTTAGGA 3′ (SEQ ID NO:61). In orderto establish that the template quality was uniform amongst all subjects,Beta Actin PCR was applied to the same amount of each template used inthe IL-17F amplification. B actin PCR included 25 cycles of PCR withsense primer, zc44779, 5′ GTGGGCCGCTCTAGGCACCA 3′ (SEQ ID NO:62) andantisense primer, zcc44776, 5′ CGGTTGGCCTTAGGGTTCAGGGGGG 3′ (SEQ IDNO:63).

All 4 mice from the DerPl sensitized, DerP1 challenged treatment group(the asthma simulation) showed robust IL-17F amplification. In contrast,weak IL-17F amplification was seen from the negative controls, including3 of 3 subjects representing the DerP1 sensitized/PBS challengedtreatment group and 5 of 5 subjects from the PBS sensitized/PBSchallenged treatment group. B actin amplification was at least as robustfor the negative controls as for the asthma-simulated subjects,demonstrating that the weak negative control IL-17F amplification wasnot due to template problems.

Example 17 COS Cell Transfection and Secretion Trap

A) Cos Cell Transfection and Secretion Trap Assays Show thatZcytoR14sR/Fc9 and IL-17F is a Receptor/Ligand Pair

A secretion trap assay was used to match the human ZcytoR14 (SEQ IDNO:2) to the human IL-17F (SE QID NO:16). The soluble ZcytoR14sR/Fc9fusion protein (Example 8) was used as a binding reagent in a secretionassay. SV40 ori containing expression vectors containing cDNA of humanIL-17B,C,D,E, and F was transiently transfected into COS cells. Thebinding of ZcytoR14sR/Fc9 to transfected COS cells was carried out usingthe secretion trap assay described below. Positive binding ofZcytoR14sR/Fc9 was only seen to human IL-17F. These results demonstratethe novel finding that human ZcytoR14 and IL-17F is a receptoriligandpair.

B) COS Cell Transfections

The COS cell transfection was performed as follows: Mix 3 ul pooled DNAand 5 ul Lipofectamine™ in 92 ul serum free DMEM media (55mg sodiumpyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5mg insulin, 1 μgselenium and 5 mg fetuin in 500 ml DMEM), incubate at room temperaturefor 30 minutes and then add 400 ul serum free DMEM media. Add this 500ul mixture onto 1.5×1O ⁵ COS cells/well plated on 12-well tissue cultureplate and incubate for 5 hours at 37° C. Add 500 ul 20% FBS DMEM media(100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 mlDMEM) and incubate overnight.

C) Secretion Trap Assay

The secretion trap was performed as follows: Media was rinsed off cellswith PBS and then fixed for 15 minutes with 1.8% Formaldehyde in PBS.Cells were then washed with TNT (0.1M Tris-HCL, 0.15M NaCl, and 0.05%Tween-20 in H₂O), and permeated with 0.1% Triton-X in PBS for 15minutes, and again washed with TNT. Cells were blockd for 1 hour withTNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NENRenaissance TSA-Direct Kit) in H₂O), and washed again with TNT. Thecells were incubated for 1 hour with lkg/mi human ZcytoR14x1sR/FC9soluble receptor fusion protein Cells were then washed with TNT. Cellswere incubated for another hour with 1:200 diluted goat-anti-humanIg-HRP (Fc specific). Again cells were washed with TNT.

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (NEN kit) and incubated for 4-6 minutes, andwashed with TNT. Cells were preserved with Vectashield Mounting Media(Vector Labs Burlingame, Calif.) diluted 1:5 in TNT. Cells werevisualized using a FITC filter on fluorescent microscope.

Example 18 Generation of Murine Anti-Human ZcytoR14 MonoclonalAntibodies

A. Immunization for Generation of Anti-ZcytoR14 Antibodies

1. Soluble ZcytoR14-muFc

Six to twelve week old intact or ZcytoR14 knockout mice are immunized byintraperitoneal injection with 25-50 ug of soluble human ZcytoR14-muFcprotein (Example 23) mixed 1:1 (v:v) with Ribi adjuvant (Sigma) on abiweekly schedule. Seven to ten days following the third immunization,blood samples were taken via retroorbital bleed, the serum harvested andevaluated for its ability to inhibit the binding of IL-17 or IL-17F toZcytoR14 in neutralization assays (e.g., described herein) and to stainZcytoR14 transfected versus untransfected 293 cells in a FACS stainingassay. Mice continued to be immunized and blood samples taken andevaluated as described above until neutralization titers reached aplateau. At that time, mice with the highest neutralization titers wereinjected intravascularly with 25-50 ug of soluble ZcytoR14-Fc protein inPBS. Three days later, the spleen and lymph nodes from these mice wereharvested and used for hybridoma generation, for example using mousemyeloma (P3-X63-Ag8.653.3.12.11) cells or other appropriate cell linesin the art, using standard methods known in the art (e.g., see Kearney,J. F. et al., J Immunol. 123:1548-50, 1979; and Lane, R. D. J ImmunolMethods 81:223-8, 1985).

2. Soluble ZcytoR14, ZcytoR14-CEE. ZcytoR14-CHIS, ZcytoR14-CFLAG

Six to twelve week old intact or ZcytoR14 knockout mice are immunized byintraperitoneal injection with 25-50 ug of soluble human ZcytoR14-CEE,ZcytoR14-CHIS, or ZcytoR14-CFLAG mixed 1:1 (v:v) with Ribi adjuvant(Sigma) on a biweekly schedule. Seven to ten days following the thirdimmunization, blood samples are taken via retroorbital bleed, the serumharvested and evaluated for its ability to inhibit the binding of IL-17or IL-17F to ZcytoR14 in neutralization assays (e.g., described herein)and to stain ZcytoR14 transfected versus untransfected 293 cells in aFACS staining assay. Mice are continued to be immunized and bloodsamples taken and evaluated as described above until neutralizationtiters reached a plateau. At that time, mice with the highestneutralization titers are injected intravascularly with 25-50 ug ofsoluble ZcytoR14, ZcytoR14-CEE, zcytor-CHIS, or ZcytoR14-CFLAG antigenprotein in PBS. Three days later, the spleen and lymph nodes from thesemice are harvested and used for hybridoma generation, for example usingmouse myeloma (P3-X63-Ag8.653.3.12.11) cells or other appropriate celllines in the art, using standard methods known in the art (e.g., seeKearney, J. F. et al., J Immunol. 123:1548-50, 1979; and Lane, R. D. JImmunol Methods 81:223-8, 1985).

3. P815 Transfectants that Express the ZcytoR14

Six to ten week old female DBA/2 mice are immunized by intraperitonealinjection of 1×10⁵ live, transfected P815 cells, for exampleP815/ZcytoR14 cells (e.g., 0.5 ml at a cell density of 2×10⁵ cells/ml).Prior to injection, the cells are maintained in the exponential growthphase. For injection the cells are harvested, washed three times withPBS and then resuspended in PBS to a density of 2×10⁵ cells/ml. In thismodel, the mice develop an ascites tumor within 2-3 weeks and progressto death by 4-6 weeks unless an immune response to the transfectedtarget antigen has been mounted. At three weeks mice with no apparentabdominal swelling (indicative of ascites) are re-immunized as above at2-3 week intervals. Seven to ten days following the second immunization,blood samples are taken via retroorbital bleed, the serum harvested andevaluated for its ability to inhibit the binding of IL-17 or IL-17F toIL-17 or ZcytoR14 in neutralization assays (e.g., described herein) andto stain ZcytoR14 transfected versus untransfected 293 cells in a FACSstaining assay. Mice continue to be immunized and blood samples takenand evaluated as described above until neutralization titers reach aplateau. At that time, the mice with the highest neutralization titersare injected intraperitonealy with 1×105 μlve, transfected P815 cells.Four days later, the spleen and lymph nodes from these mice areharvested and used for hybridoma generation, for example using mousemyeloma (P3-X63-Ag8.653.3.12.11) cells or other appropriate cell linesin the art, using standard methods known in the art (e.g., see Kearney,J. F. et al., supra.; and Lane, R. D. supra.).

An alternative to the above immunization scheme with live, transfectedP815 cells involves intraperitoneal injection of 1-5×10⁶ irradiated,transfected cells every 2-3 weeks. In this approach, no animals developand die of ascites. Instead, animals are monitored for a neutralizingimmune response to ZcytoR14 in their serum as outlined above, startingwith a bleed after the second immunization. Once neutralization titershave reached a maximal level, the mice with highest titers are given apre-fusion, intraperitoneal injection of 5×10⁶ irradiated cells and fourdays later, the spleen and lymph nodes from these mice are harvested andused for hybridoma generation, for example using mouse myeloma(P3-X63-Ag8.653.3.12.11) cells or other appropriate cell lines in theart, using standard methods known in the art (e.g., see Kearney, J. F.et al., supra.; and Lane, R. D. supra.).

B. Screening the Hybridoma Fusions for Antibodies that bind ZcytoR14 andInhibit the Binding of IL-17 or IL-17F to ZcytoR14

Three different primary screens are performed on the hybridomasupernatants at 8-10 days post-fusion. For the first assay, antibodiesin supernatants were tested for their ability to bind to plate boundsoluble human ZcytoR14, ZcytoR14-muFc, ZcytoR14-CEE, ZcytoR14-CHIS, orZcytoR14-CFLAG protein by ELISA using HRP-conjugated goat anti-mousekappa and anti-lambda light chain second step reagents to identify boundmouse antibodies. To demonstrate specificity for the ZcytoR14 portion ofthe ZcytoR14 fusion proteins, positive supernatants in the initial assaywere evaluated on an irrelevant protein fused to the same murine Fcregion (mG2a), EE sequence, HIS sequence, or FLAG sequence. Antibody inthose supernatants that bound to ZcytoR14-fusion protein and not theirrelevant muFc or other proteins containing fusion protein sequencewere deemed to be specific for ZcytoR14. For the second assay,antibodies in all hybridoma supernatants were evaluated by ELISA fortheir ability to inhibit the binding of biotinylated human IL-17 orbiotinylated human IL-17F to plate bound ZcytoR14-muFc orZcytoR14-fusion proteins.

All supernatants containing antibodies that bound specifically toZcytoR14, whether they inhibited the binding of IL-17 or IL-17F toZcytoR14 or not in the ELISA assay, were subsequently tested for theirability to inhibit the binding of IL-17 or IL-17F to ZcytoR14transfected Baf3 or BHK cells or normal human bronchial epithelialcells. All supernatants that were neutralization positive in either theIL-17 or IL-17F inhibition assays or both the IL-17 and IL-17Finhibition assays were subsequently evaluated for their ability to stainZcytoR14 transfected versus non-transfected Baf3 or BHK cells by FACSanalysis. This analysis was designed to confirm that inhibition of IL-17or IL-17F binding to ZcytoR14, was indeed due to an antibody thatspecifically binds the ZcytoR14 receptor. Additionally, since the FACSanalysis was performed with an anti-IgG second step reagent, specificFACS positive results indicate that the neutralizing antibody was likelyto be of the IgG class. By these means, a master well was identifiedthat bound ZcytoR14 in the plate bound ELISA, inhibited the binding ofIL-17 or IL-17F to ZcytoR14 in the ELISA based inhibition assay, blockedthe interaction of IL-17 and IL-17F with ZcytoR14 transfected Baf3 orBHK cells, respectively, and was strongly positive for the staining ofZcytoR14 transfected Baf3 or BHK cells with an anti-mouse IgG secondstep reagent.

The third assay consists of primary human bronchial epithelial cellswhich express ZcytoR14 and can be induced to secrete IL-8 or IL-6 inresponse to IL-17F treatment. The specific monoclonal antibody isassayed by its ability to inhibit the IL-17 or IL-17F stimulated IL-8 orIL-6 production by these cells. IL-8 and IL-6 production is assayed inresponse to IL-17 or IL-17F as described herein.

Alternatively, the monoclonal antibody; anti-ZcytoR14, mediatedinhibition of IL-17 or IL-17F induced luciferase production in NIH 3T3or other ZcytoR14 containing cells can be used with or in place of oneof the bioactivity neutralization assays noted above. The NFkB mediatedluciferase assay in NIH 3T3 cells is described herein.

C) Cloning Anti-ZcvtoR14 Specific Antibody Producing Hybridomas

Hybridoma cell lines producing a specific anti-ZcytoR14 mnAb thatcross-neutralized the binding of IL-17 and IL-17F to appropriatelytransfected BaF3 or BHK cells are cloned by a standard low-densitydilution (less than 1 cell per well) approach. Approximately 5-7 daysafter plating, the clones are screened by ELISA on, for example, platebound human ZcytoR14-muFc followed by a retest of positive wells byELISA on irrelevant muFc containing fusion protein as described above.Selected clones, whose supernatants bind to ZcytoR14-muFc and not theirrelevant muFc containing fusion protein, are further confirmed forspecific antibody activity by repeating both neutralization assays aswell as the FACS analysis. All selected ZcytoR14 antibody positiveclones are cloned a minimum of two times to help insure clonality and toassess stability of antibody production. Further rounds of cloning areperformed and screened as described until, preferably, at least 95% ofthe resulting clones were positive for neutralizing anti-ZcytoR14antibody production.

D) Biochemical Characterization of the Molecule Recognized byAnti-ZcytoR14 mAbs

Biochemical confirmation that the target molecule, ZcytoR14, recognizedby the putative anti-ZcytoR14 mAbs is indeed ZcytoR14 are performed bystandard immunoprecipitation followed by SDS-PAGE analysis or westernblotting procedures, both employing soluble membrane preparations fromZcytoR14 transfected versus untransfected Baf3 or BHK cells. Moreover,soluble membrane preparations of non-transfected cell lines that expressZcytoR14 are used show that the mAbs recognize the native receptor chainas well as the transfected one. Alternatively, the mAbs are tested fortheir ability to specifically immunoprecipitate or western blot thesoluble ZcytoR14-muFc protein.

Example 19 Neutralization of Human ZcytoR14 by Sera from Mice Injectedwith P815 Cells Transfected with Human ZcytoR14

Using a cell based neutralization assay, serum from mice injected withlive human ZcytoR14 transfected P815 cells (Example 17) is added as aserial dilution at 1%, 0.5%, 0.25%, 0.13%, 0.06%, 0.03%, 0.02%, and 0%.The assay plates are incubated at 37 □C, 5% CO₂ for 4 days at which timeAlamar Blue (Accumed, Chicago, Ill.) is added at 20 μl/well. Plates areagain incubated at 37 □C, 5% CO₂ for 16 hours. Results showed that serumfrom four of the animals could neutralize signaling of both huIL-17 andhuIL-17F through human ZcytoR14.

Results such as these provide additional evidence that effectivelyblocking ZcytoR14 by binding, blocking, inhibiting, reducing,antagonizing or neutralizing IL-17 or IL-17F activity (individually ortogether), for example via a neutralizing monoclonal antibody toZcytoR14 of the present invention, could be advantageous in reducing theeffects of IL-17 and IL-17F (alone or together) in vivo and may reduceIL-17 and/or IL-17F-induced inflammation, such as that seen in, forexample in psoriasis, IBD, colitis, chronic obstructive pulmonarydisease, cystic fibrosis or other inflammatory diseases induced byIL-17, and or IL-17F including IBD, arthritis, asthma, psoriaticarthritis, colitis, inflammatory skin conditions, and atopic dermatitis.

Example 20 Pharmacokinetics of an Anti-human ZcytoR14 MonoclonalAntibody

The test monoclonal antibody, anti-human ZcytoR14 mAb, is provided in,for example, 3×3 mL aliquots at a concentration of approximately 1 mg/mL(determined by UV Absorbance at 280 nM) and was stored at −80° C. untiluse. The vehicle is 1× PBS (50 mM NaPO4, 109 mM NaCl), pH 7.3. The mAbis thawed at room temperature before use and aliquots 1 and 2 are usedas provided for the 100 μg IV and SC dosing groups, respectively. Halfof aliquot 3 is diluted 1:2 in 1× PBS for the 50 μg SC dose group andthe second half of aliquot 3 is diluted 1:10 in 1× PBS for the 10 μg SCdose group. Female SCID mice (n=96) are obtained from Charles RiverLabs. Animals are checked for health on arrival and group-housed (3animals per cage). The mice are 12 weeks old with an average body weightof approximately 22 g at the beginning of the study.

A) Dosing Protocol

Female SCID mice (n=24/dose group) are randomly placed into four dosinggroups (Table 5). Group 1 was administered the anti-human ZcytoR14 TnAbvia IV injection of approximately 93 μL in a tail vein and Groups 2, 3,and 4 are administered the mAb via SC injection of approximately 93 μLin the scruff of the neck.

B) Sample Collection

Prior to blood collection, mice were fully anesthetized with halothaneor isofluorane. Blood samples were collected via cardiac stick for alltime points except the 168 hr timepoint (collected via eye bleed and thesame animals were bled again at the 504 hr timepoint via cardiac stick).Blood was collected into serum separator tubes and allowed to clot for15 minutes. Samples were subsequently centrifuged for 3 minutes at14,000 rpm. Following centrifugation, aliquots of 125-150 uL weredispensed into labeled eppendorf tubes and immediately stored at −80° C.until analysis. TABLE 5 Group # Dose (ROA) Animals PK Timepoints 1 100μg (IV) 3 mice/ 0.25, 1, 4, 8, 24, 72, 168, 336 timepoint* and 504 hr 2100 μg (SC) 3 mice/ 0.25, 1, 4, 8, 24, 72, 168, 336 timepoint* and 504hr 3 50 μg (SC) 3 mice/ 0.25, 1, 4, 8, 24, 72, 168, 336 timepoint* and504 hr 4 10 μg (SC) 3 mice/ 0.25, 1, 4, 8, 24, 72, 168, 336 timepoint*and 504 hr*The same animals were used for the 168 and 504 hr timepoints.C) Quantification of Serum Anti-Human ZcytoR14 mAb Concentrations byELISA

An Enzyme Linked Immunosorbant Assay (ELISA) is developed and qualifiedto analyze mouse serum samples from animals dosed with anti-ZcytoR14 mAbduring pharmacokinetic studies. This assay is designed to take advantageof a commercially available secondary antibody and colorimetricdetection using TMB. The dilutions used for the standard curve weremodified to improve the definition of the linear portion of the standardcurve. A standard curve in the range of 100 ng/mL to 0.231 ng/mL with2-fold dilutions allows for quantitation of the mouse serum samples. QCsamples are diluted to 1:100, 1:1000 and 1:10000 in 10% SCID mouse serumand back calculated from the standard curve.

D) Pharmacokinetic Analysis

Serum concentration versus time data are downloaded into WinNonlinProfessional 4.0 software (Pharsight, Inc.; Cary, N.C.) forpharmacokinetic analysis. Noncompartmental analysis is used to determinepharmacokinetic parameters based on the mean data at each time point.

Example 21 Neutralization of IL-17 and IL-17F Activity by a Anti-HumanZcytoR14 Monoclonal Antibody

Using a cell-based neutralization assay, a purified mouse anti-humanZcytoR14 monoclonal antibody is added as a serial dilution, for example,at 10 μg/ml, 51 μg/ml, 2.5 μg/ml, 1.25 μg/ml, 625 ng/ml, 313 ng/ml, 156ng/ml and 78 ng/ml. The assay plates are incubated at 37° C., 5% CO₂ for4 days at which time Alarnar Blue (Accumed, Chicago, Ill.) is added at20 μl/well. Plates are again incubated at 37 □C, 5% CO₂ for 16 hours.This assay is able to demonstrate that the purified anti-human ZcytoR14monoclonal antibody is able neutralize signaling of both huIL-17 andhuEL-17F through human ZcytoR14. For highly effective antibodies, whenused at approx. 10 μg/ml concentration, the antibody completelyneutralizes proliferation induced by huIL-17 or huIL-17F, with theinhibition of proliferation decreasing in a dose dependent fashion atthe lower concentrations. An isotype-matched negative control mouse mAb,tested at the concentrations described above, is exected to provide noinhibition of proliferation of either cytokine. These results are ableto further demonstrate that monoclonal antibodies to ZcytoR14 couldindeed antagonize the activity of the pro-inflammatory ligands, IL-17and IL-17F at low concentrations.

Example 22 IL-17A Induces Elevated Levels of IFN-Gamma and TNF-Alpha inHuman Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMC) are purified by ficolldensity gradient centrifugation and then incubated overnight at 37° C.in media alone, 50 ng/ml anti-human CD3 antibody, or the combination of50 ng/ml anti-human CD3 antibody plus 1 □g/ml anti-human CD28 antibody.Replicate cultures for each of these conditions are set up and are givenno cytokine, 25 ng/ml human IL-17A, or 25 ng/ml human IL-17F. After24-hour incubations, supernatants from each culture are harvested andassayed for cytokine content using B-D Bioscience's human Th1/Th2Cytometric Bead Array (CBA). We found that cultures that had beenstimulated with either anti-CD3 or anti-CD3 plus anti-CD28 and had beensupplemented with IL-17A contained significantly elevated levels ofIFN-gamma and TNF-alpha (3-5-fold elevation of each) over cultures withno cytokine added or those that received IL-17F. Cultures in which noanti-CD3 stimulation was added did not show significant changes incytokine levels. In addition, IL-17A addition induced no significantchanges in other cytokines assayed for with the CBA including IL-2,IL-4, IL-S, and IL-10. This data indicates that IL-17A, but not IL-17F,can augment the production of IFN-gamma and TNF-alpha in PBMC culturesstimulated with anti-CD3 or anti-CD3 plus anti-CD28.

Example 23 ZcytoR14-Fc Decreases Disease Incidence and Progression inMouse Collagen Induced Arthritis (CIA) Model

A) Mouse Collagen Induced Arthritis (CIA) Model

Ten week old male DBA/1J mice (Jackson Labs) are divided into 3 groupsof 13 mice/group. On day-21, animals are given an intradermal tailinjection of 50-100 μl of 1 mg/ml chick Type II collagen formulated inComplete Freund's Adjuvant (prepared by Chondrex, Redmond, Wash.), andthree weeks later on Day 0 they are given the same injection exceptprepared in Incomplete Freund's Adjuvant. ZcytoR14-Fc is administered asan intraperitoneal injection 3 times a week for 4 weeks, at differenttime points ranging from Day 0, to a day in which the majority of miceexhibit moderate symptoms of disease. Groups receive either 10 or 100 μgof ZcytoR14-Fc per animal per dose, and control groups receive thevehicle control, PBS (Life Technologies, Rockville, Md.). Animals beginto show symptoms of arthritis following the second collagen injection,with most animals developing inflammation within 1.5-3 weeks. The extentof disease is evaluated in each paw by using a caliper to measure pawthickness, and by assigning a clinical score (0-3) to each paw:0=Normal, 0.5=Toe(s) inflamed, 1=Mild paw inflammation, 2=Moderate pawinflammation, and 3=Severe paw inflammation as detailed below.

B) Monitoring Disease

Animals can begin to show signs of paw inflammation soon after thesecond collagen injection, and some animals may even begin to have signsof toe inflammation prior to the second collagen injection. Most animalsdevelop arthritis within 1.5-3 weeks of the boost injection, but somemay require a longer period of time. Incidence of disease in this modelis typically 95-100%, and 0-2 non-responders (determined after 6 weeksof observation) are typically seen in a study using 40 animals. Notethat as inflammation begins, a common transient occurrence of variablelow-grade paw or toe inflammation can occur. For this reason, an animalis not considered to have established disease until marked, persistentpaw swelling has developed.

All animals are observed daily to assess the status of the disease intheir paws, which is done by assigning a qualitative clinical score toeach of the paws. Every day, each animal has its 4 paws scored accordingto its state of clinical disease. To determine the clinical score, thepaw can be thought of as having 3 zones, the toes, the paw itself (manusor pes), and the wrist or ankle joint. The extent and severity of theinflammation relative to these zones is noted including: observation ofeach toe for swelling; torn nails or redness of toes; notation of anyevidence of edema or redness in any of the paws; notation of any loss offine anatomic demarcation of tendons or bones; evaluation of the wristor ankle for any edema or redness; and notation if the inflammationextends proximally up the leg. A paw score of 1, 2, or 3 is based firston the overall impression of severity, and second on how many zones areinvolved. The scale used for clinical scoring is shown below.

C) Clinical Score

0=Normal

0.5=One or more toes involved, but only the toes are inflamed

1=mild inflammation involving the paw (1 zone), and may include a toe ortoes

2=moderate inflammation in the paw and may include some of the toesand/or the wrist/ankle (2 zones)

3=severe inflammation in the paw, wrist/ankle, and some or all of thetoes (3 zones)

Established disease is defined as a qualitative score of pawinflammation ranking 2 or more, that persists for two days in a row.Once established disease is present, the date is recorded and designatedas that animal's first day with “established disease”.

Blood is collected throughout the experiment to monitor serum levels ofanti-collagen antibodies, as well as serum immunoglobulin and cytokinelevels. Serum anti-collagen antibodies correlate well with severity ofdisease. Animals are euthanized on Day 21, and blood collected for serumand CBC's. From each animal, one affected paw is collected in 10%NBF forhistology and one is frozen in liquid nitrogen and stored at −80° C. formRNA analysis. Also, ½ spleen, ½ thymus, ½ mesenteric lymph node, oneliver lobe and the left kidney are collected in RNAlater for RNAanalysis, and 0.½ spleen, 1/2 thymus, ½ mesenteric lymph node, theremaining liver, and the right kidney are collected in 10% NBF forhistology. Serum is collected and frozen at −80° C. for immunoglobulinand cytokine assays.

Groups of mice receiving ZcytoR14-Fc at all time points arecharacterized by a delay in the onset and/or progression of pawinflammation. These results indicate that ZcytoR14 can reduceinflammation, as well as disease incidence and progression associatedwith this model. These results are further supported by the observationthat ZcytoR14-Fc resulted in decreased levels of serum TNFa, IL-1b, andanti-collagen antibodies.

Example 24 Stable Over-Expression of ZcytoR14 in the Murine Assay CellLine, Nih3t3/kz142.8 Expressing the ap1/nfkb Transcription Factor

The murine nih3t3/kz142.8 assay cell line was transfected with a humanzcytor14x1 (SEQ ID NO:2) in an expression vector with a methotrexateresistance gene (dihydrofolate reductase,DHFR) This transfection wasperformed using a commercially available kit and the manufacturer'srecommendations. (Mirus, Madison, Wis. Cat. #MIR218) Cells were placedin 1 μM mtx amended growth medium to select for the expression vectorcontaining the human zcytor14X1 transgene. After selection a humanzcytor14x1 transfection pool was generated, and callednih3t3/kz142.8/hcytor14x1.

A) Luciferase Assay Using the nih3t3/kz142.8 Assay Cell Line

Since nih3t3/kz142.8 has a stable kz142 reporter, there is no need foradenovirus infection to add this reporter. Thus the luciferase assayprotocol was shorted and done the following way:

1. Cell Plating

nih3t3/kz142.8 cells were plated at 5000 cells/well in solid white, cellculture coated 96 well plates, (Cat. #3917. Costar) using DMEM/10% FBS,containing glutamine and amended with pyruvate and cultured overnight at37 oC and 5% C02. On this second day, the plating media was removed andexchanged for DMEM/1% FBS, containing glutamine and amended withpyruvate and cultured overnight at 37 oC and 5% C02.

2. Luciferase Assay Measuring IL-17A and F Activation of the Stablekz142 Reporter

Following the overnight incubation in the 1% fbs, DMEM media, humanIL-17A,and IL-17F ligand dilutions were made in serum free media,amended with BSA to a 0.28% level. After adding the ligand dilutions,cells were incubated at 37 oC and 5% C02 for 4 hours, after which themedia was removed, cells lysed for 15 minutes and mean fluorescenceintensity (MFI) measured using the luciferase assay system and reagents,(Cat.#e1531 Promega. Madison, Wis.) and a Microplate luminometer.Activity was detected for both ligands at concentrations ranging from0.1-1000 ng/ml. The nih3t3/kz142.8/hcytor14x1 transfection pool showedsimilar activity for the murine IL-17A ligand as did the parental cellline. (example 14) However, the cytor14x1 transfectant pool showed anelevated responsiveness to human IL-17A and F treatments, even whenthese ligand concentrations were as low as 20 femptograms. The fact thatthe mIL-17A signaling is comparable to that in the parental cell line(examplel4) suggests that there isn't a general, non-specific problemwith human ZcytoR14-expressing cells and that the murine IL-17A isprobably signaling through the endogenous murine nih3t3 cell IL-17R orZcytoR14 receptor. Thus, the fact that human IL-17A and IL-17F cause anelevation of MFI at such low ligand concentrations may indicate aspecific hyper-responsiveness of the cells to those ligands, which ismediated through the over-expressed human ZcytoR14 receptor.

This result has significant clinical and biological ramifications andutility. For example, physiological situations could cause localup-regulation of the ZcytoR14 receptors which could then make theseareas hyper-responsive to IL-17A and IL-17F, resulting in biologicalactivation at much lower ligand concentrations than those suggestedwithout ZcytoR14 over-expression. Thus, far lower soluble receptorlevels may be sufficient to antagonize these hypothetically lower ligandconcentrations, than previously thought or recognized by those in thefield.

Example 25 Antagonists to IL-17F and IL-17A activity Decrease DiseaseIncidence and Progression in an Inflammatory Bowel Disease (IBD) Model

This model is designed to show that cultured intestinal tissue frompatients with IBD produce higher levels of inflammatory mediatorscompared to tissue from healthy controls. This enhanced production ofinflammatory mediators (including but not limited to IL-1b, IL-4, IL-5,IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A and F, IL-18, IL-23, TNF-a,IFN-g, MIP family members, MCP-1, G- and GM-CSF, etc.) contributes tothe symptoms and pathology associated with IBDs such as Crohn's disease(CD) and ulcerative colitis (UC) by way of their effect(s) on activatinginflammatory pathways and downstream effector cells. These pathways andcomponents then lead to tissue and cell damage/destruction observed invivo. Therefore, this model can simulate this enhanced inflammatorymediator aspect of IBD. Furthermore, when intestinal tissue from healthycontrols or from human intestinal epithelial cell (EEC) lines iscultured in the presence of these inflammatory components, inflammatorypathway signaling can be observed, as well as evidence of tissue andcell damage.

Therapeutics that would be efficacious in human IBD in vivo would workin the above ex vivo or IEC models by inhibiting and/or neutralizing theproduction and/or presence of inflammatory mediators.

In this model, human intestinal tissue is collected from patients withIBD or from healthy controls undergoing intestinal biopsy, re-sectioningor from post-mortem tissue collection, and processed using amodification of Alexakis et al (Gut 53:85-90; 2004). Under asepticconditions, samples are gently cleaned with copious amounts of PBS,followed by culturing of minced sections of tissue, in the presence ofcomplete tissue culture media (plus antibiotics to prevent bacterialovergrowth). Samples from the same pool of minced tissue are treatedwith one of the following: vehicle (PBS); recombinant human (rh) IL-17A;rhIL-17F; or rhIL-17A+rhIL-17F. In addition, these are treated with orwithout an antagonist of either IL-17A or IL-17F, alone or incombination (such as a soluble ZcytoR14). This experimental protocol isfollowed for studies with human IEC lines, with the exception that cellsare passaged from existing stocks. After varying times in culture (from1 h to several days), supernatants are collected and analyzed for levelsof inflammatory mediators, including those listed above. In samples frompatients with IBD or in samples treated with rhIL-17A and/or F, levelsof inflammatory cytokines and chemokines are elevated compared tountreated healthy control tissue samples. The addition of antagonists toIL-17F and/or IL-17A activity, such as ZcytoR14 soluble receptors andantibodies thereto including the anti-human-ZcytoR14 monoclonal andneutralizing antibodies of the present invention markedly reduces theproduction of inflammatory mediators, and thus, would expect to beefficacious in human IBD.

Example 26 Antagonists to IL-17F and IL-17A activity Decrease DiseaseIncidence and Progression in a Multiple Sclerosis (MS) Model

Multiple sclerosis (MS) is a complex disease that is thought to bemediated by a number of factors, including the presence of lymphocyticand mononuclear cell inflammatory infiltrates and demyelinationthroughout the CNS. Microglia are macrophage-like cells that populatethe central nervous system (CNS) and become activated upon injury orinfection. Microglia have been implicated as playing critical roles invarious CNS diseases including MS, and may be used to study mechanism(s)of initiation, progression, and therapy of the disease (Nagai et al.Neurobiol Dis 8:1057-1068; 2001; Olson et al. J Neurosci Methods128:33-43; 2003). Immortalized human microglial cell lines and/orestablished human astroglia cell lines can, therefore, be used to studysome of the effects of inflammatory mediators on these cell types andtheir potential for neutralization. Inflammatory mediators (includingbut not limited to IL-1b, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A andF, IL-18, IL-23, TNF-a, IFN-g, MIP family members, RANTES, IP-10, MCP-1,G- and GM-CSF, etc.) can contribute to the symptoms and pathologyassociated with MS by way of their effect(s) on activating inflammatorypathways and downstream effector cells.

In order to evaluate the pro-inflammatory actions of IL-17A and IL-17F,and the ability of an antagonist to IL-17F and/or IL-17A activity, suchas ZcytoR14 soluble receptors and antibodies thereto including theanti-human-ZcytoR14 monoclonal and neutralizing antibodies of thepresent invention to neutralize or decrease these effects, culturedglial cells are treated with one of the following: vehicle; rhIL-17A;rhIL-17F; rhIL-17A+IL-17F. In addition, these are treated with orwithout an antagonist of either IL-17A or IL-17F, alone or incombination (such as a soluble ZcytoR14). After varying times in culture(from 1 h to several days), supernatants and cells are collected andanalyzed for levels and/or expression of inflammatory mediators,including those listed above. Levels of inflammatory cytokines andchemokines are elevated in the presence of rhIL-17A and/or IL-17Fcompared to cultures treated with vehicle alone. The addition ofantagonists to IL-17F and/or IL-17A activity, such as ZcytoR14 solublereceptors and antibodies thereto including the anti-human-ZcytoR14monoclonal and neutralizing antibodies of the present invention markedlyreduces the production and expression of inflammatory mediators, andthus, would expect to be efficacious in inflammatory aspects associatedwith human MS.

Example 27 Antagonists to IL-17F and IL-17A activity Decrease DiseaseIncidence and Progression in a Rheumatoid Arthritis (RA) andOsteoarthritis (OA) Model

This model is designed to show that human synovial cultures (includingsynovial macrophages, synovial fibroblasts, and articular chondrocytes)and explants from patients with RA and OA produce higher levels ofinflammatory mediators compared to cultures/explants from healthycontrols. This enhanced production of inflammatory mediators (includingbut not limited to oncostatin M, IL-1b, IL-6, IL-8, IL-12, IL-15, IL-17A and F, IL-18, IL-23, TNF-a, IFN-g, IP-10, RANTES, RANKL, MIP familymembers, MCP-1, G- and GM-CSF, nitric oxide, etc.) contributes to thesymptoms and pathology associated with RA and OA by way of theireffect(s) on activating inflammatory pathways and downstream effectorcells. These pathways and components then lead to inflammatoryinfiltrates, cartilage and matrix loss/destruction, bone loss, andupregulation of prostaglandins and cyclooxygenases. Therefore, thismodel can simulate the destructive inflammatory aspects of RA and OA inin vitro and ex vivo experiments. Furthermore, when explants andsynovial cultures from healthy controls are cultured in the presence ofseveral of these inflammatory components (e.g. oncostatin M, TNF-a,IL-1b, IL-6, IL-17A and F, IL-15, etc.), inflammatory pathway signalingcan be observed. Therapeutics that would be efficacious in human RA invivo would work in the above in vitro and ex vivo models by inhibitingand/or neutralizing the production and/or presence of inflammatorymediators.

In this model, human synovial explants are collected from patients withRA, OA, or from healthy controls undergoing joint replacement or frompost-mortem tissue collection, and processed using a modification ofWooley and Tetlow (Arthritis Res 2: 65-70; 2000) and van't Hof et al(Rheumatology 39:1004-1008; 2000). Cultures of synovial fibroblasts,synovial macrophages and articular chondrocytes are also studied.Replicate samples are treated with one of the following: vehicle (PBS);recombinant human (rh) IL-17A; rhIL-17F; or rhIL-17A+rhIL-17F, and somesamples contain various combinations of oncostatin M, TNF-a, IL-1b,IL-6, IL-17A, IL-17F, and IL-15. In addition, these are treated with orwithout an antagonist to IL-17F and/or IL-17A activity, such as ZcytoR14soluble receptors and antibodies thereto including theanti-human-ZcytoR14 monoclonal and neutralizing antibodies of thepresent invention. After varying time of culture (from 1 h to severaldays), supernatants are collected and analyzed for levels ofinflammatory mediators, including those listed above. In samples frompatients with RA or OA, or in samples treated with rhIL-17A and/or F(either alone or in combination with other inflammatory cytokines),levels of inflammatory cytokines and chemokines are elevated compared tountreated healthy control explants or in untreated cell cultures. Theaddition of antagonists to IL-17F and/or IL-17A activity, such asZcytoR14 soluble receptors and antibodies thereto including theanti-human-ZcytoR14 monoclonal and neutralizing antibodies of thepresent invention markedly reduces the production of inflammatorymediators, and thus, would expect to be efficacious in human RA and OA.

Example 28 IL-17A and IL-17F Functional Responses

NIH-3T3/KZ142 cells were stably transfected with human zcytoR14x1 (SEQID NO:1) and mouse zcytoR14x1 (SEQ ID NO:25). As described above, eachline was treated for 7 and 15 minutes with a dose response of IL-1 7A,IL-17F, murine IL-17F, and appropriate controls. Both IL-17A and IL-17Fgave a dose dependent response in phosphorylated IκB-α and p38 MAPKtranscription factors when ZcytoR14x1 (SEQ ID NO:1) was transfected,approximately 30% greater then the inherent signaling from the controlline. IL-17A and IL-17F gave no increase in signaling when the murimeZcytoR14x1 (SEQ ID NO:25) was transfected. Murine IL-17F gave noincrease in signaling for either human or murine ZcytoR14x1.

Example 29 IL-17A, IL-17F, IL-17R and ZcytoR14 Expression in MurineDisease Models

Four murine models of disease (asthma, DSS colitis, atopic dermatitisand experimental allergic encephalomyelitis) were analyzed using knowtechniques for the expression of IL-17A, IL-17F, IL-17R and ZcytoR14.

In the asthma model, IL-17A and IL-17F are expressed at very low toundetectable levels in lung, spleen, lung draining lymph nodes and lunginfiltrating cells in diseased and non-diseased mice. Zcytor14 messagewas found to be more highly expressed in lung compared to spleen andlymph node but was not regulated with disease. IL-17R was more highlyexpressed in spleen and lung draining lymph node compared to lung butwas also not regulated with disease.

Contrary to the asthma model, IL-17A and IL-17F were highly up-regulatedin diseased but not normal mice in the DSS-colitis model in bothproximal and distal colon. Neither cytokine was significantlyup-regulated in the mesenteric lymph node. Further, it was found thatup-regulation of both cytokines in the context of acute DSS-inducedcolitis and not in chronic DSS-induced colitis. IL-17R was found to beprominently expressed in mesenteric lymph nodes as compared to proximaland distal colon, but was not regulated with disease. In contrast,ZcytoR14 was more highly expressed in proximal distal colon tissuecompared to mesenteric lymph nodes. ZcytoR14 expression was also notregulated with disease.

In atopic dermatitis, IL-17A mRNA was not detectable. IL-17F was foundto be expressed in both skin and skin-raining lymph nodes but did notappear to be significantly regulated with disease. IL-17R mRNA was morehighly expressed in skin-draining lymph nodes as compared to skin butwas not regulated with disease. Zcytor14 was more highly expressed inskin compared to skin-draining lymph nodes but was also not regulatedwith disease.

In experimental allergic encephalomyelitis, both IL-17A and IL-17Fappeared to up-regulated in spinal chord in diseased but not healthymice. IL-17F may have been more highly expressed in lymph nodes comparedto spinal cord but expression in the lymph nodes was not regulated withdisease. However, overall levels of expression in these tissues wasquite low. IL-17R was more highly expressed in lymph node tissuecompared to brain and spinal cord. Zcytor14 was not tested.

In short, IL-17A and IL-17F expression appears to be regulated withdisease in the context of the DSS-induced colitis and experimentalallergic encephalomyelitis models but apparently not for asthma oratopic dermatitis. IL-17R and zcytor14 expression does not appear to beregulated with disease but IL-17R expression appears to be enriched inlymphoid tissues while zcytor14 expression appears to be enriched innon-lymphoid tissues.

Example 30 ZcytoR14 is a Mediator of Activation to Both IL-17A andIL-17F

The murine nih3t3/kz142.8 assay cell line was transfected with a humanZcytoR14X1 (SEQ ID NO:2) in an expression vector with a methotrexateresistance gene. (dihydrofolate reductase,DHFR) Human IL-17RA (SEQ IDNO:21) was similarly tranfected into this cell line. Transfections wereperformed using a commercially available kit and the manufacturer'srecommendations. (Mirus, Madison, Wis. Cat. #MIR218) Cells were placedin 1 μM mtx amended growth medium to select for the expression vectorcontaining the expression constructs. After selection transfection poolswere generated, and called nih3t3/kz 142.8/hcytor14X 1 andnih3t3/kz142.8/IL-17R.

A) Luciferase Assay Using the nih3t3/kz142.8-Based Cell Lines.

Since nih3t3/kz142.8 based cell lines have stable ap1/nfkb reporters(kz142), there is no need for adenovirus infection to add this reporter.Thus the luciferase assay protocol was shorted and done the followingway:

1. Cell Plating

Cells were plated at 5000 cells/well in solid white, cell culture coated96 well plates, (Cat. #3917. Costar) using DMEM/10% FBS, containingglutamine and amended with pyruvate and cultured overnight at 37 oC and5% C02. On this second day, the plating media was removed and exchangedfor DMEM/1% FBS, containing glutamine and amended with pyruvate andcultured overnight at 37 oC and 5% C02.

2. Luciferase Assay Measuring IL-17A and F Activation of the Stablekz142 Reporter

Following the overnight incubation in the 1% fbs, DMEM media, humanIL-17A,and IL-17F ligand dilutions were made in serum free media,amended with BSA to a 0.28% level. After adding the ligand dilutions,cells were incubated at 37 oC and 5% C02 for 4 hours, after which themedia was removed, cells lysed for 15 minutes and mean fluorescenceintensity (MFI) measured using the luciferase assay system and reagents,(Cat.#e1531 Promega. Madison, Wis.) and a Microplate luminometer.Activity was detected for both ligands at concentrations ranging from0.1-100 ng/ml.

The EC_(50s) discussed below are averages of at least 4 experiments. Thenih3t3/kz142.8/hcytor14x1 transfection pool showed similar activity forthe murine IL-17A ligand as did the parental cell line, with an EC₅₀ ofabout 4 ng/ml. (example 14) The fact that the mIL-17A signaling in thehcytor14x1 recombinant line is comparable to that in the parental cellline (example14) suggests that murine IL-17A is probably signalingthrough the endogenous murine nih3t3 cell IL-17RA or zcytor14 receptorsand does not activate the cells through hcytor14X1. However, thehzcytor14X1 transfectant pool showed an elevated responsiveness to humanIL-17A treatment, with an EC₅₀ of 0.41 ng/ml Vs 2.8 ng/ml (averages of 4experiments) in the parental line (a 6.8 fold more potent EC₅₀ in therecombinant line) In addition, the hIL-17RCX1 recombinant line had anenhanced responsiveness to hIL-17F, with an EC₅₀ of 0.61 ng/ml in therecombinant line Vs 10 ng/ml in the parental line. (a 17 fold morepotent EC₅₀ in the recombinant line). The increased potency to hIL-17Aand F in the hIL-17RCX1 line is consistent with human zcytor14X1 being ahigh affinity receptor for both human IL-17A and IL-17F. In contrast,the hIL-17RA recombinant line had enhanced sensitivity only to hIL-17A,with an EC₅₀ of 0.6 ng/ml vs 2.8 ng/ml for the parental line. There wasnot an enhancement of the hIL-17F EC₅₀ in the hIL-17RA recombinant line,with an IL-17F EC₅₀ of 12.4 ng/ml vs 8.9 ng/ml in the parental line.

This result is significant because it specifically implicateshzcytor14X1 as a mediator of activation to both hIL-17A and hIL-17F andsuggests that hIL-17RA mediates signaling only to hIL-17A activation andnot hIL-17F.

Example 31 Intravenous Admninistration of IL-17A and IL-17F

To determine the effect of i.v. delivery of murine or human IL-17A orIL-17F on complete blood counts (CBC) and serum cytokines/chemokines inBALB/c mice at various time points.

I.V. administration of 1 ug mIL-17A resulted in an approximate 2-foldincrease in circulating neutrophils (by CBC) and approximate 10-foldincrease in serum KC and MCP-1 (by Luminex) 1-2 h followingadministration; similar results in these chemokines were observed with 5ug hIL-17A. Blood monocyte levels were also significantly increased inmice treated with 1 ug mIL-17A (showed the greatest increase), 5 ughIL-17A or 5 ug hIL-17F at the 2 h timepoint. I.V. administration of mand hIL-17F resulted in marked increases in serum IL-15 (by Luminex) atthe 1 and 2 h time points, and small increases in serum KC and MCP-1 atthese same timepoints.

Example 32 Neutralization of i.v. Administered IL-17A and IL-17F

To neutralize the i.v. IL-17A and IL-17F-mediated increases in cytokinesand chemokines with i.p. soluble receptors (mIL-17RA:Fc for murineligands; soluble human ZcytoR14 for human ligands). Female BALB/c micewere administered by i.p. injection either PBS, 100 ug mIL-17RA:Fc, or100 ug soluble human ZcytoR14 three hours prior to receiving by i.v.tail injection: PBS; 2 ug of either mIL-17A, mIL-17F, or 2 ug of bothmIL-17A and F (for mice that received mIL-17RA:Fc); or 2 ug of eitherhIL-17A, hIL-17F, or 2 ug of both hIL-17A and F (for mice that receivedsoluble human ZcytoR14). Serum was collected 1 h following ligandadministration and analyzed for a small number of serum cytokines andchemokines.

Mice pretreated with i.p. soluble receptor had marked reductions inIL-17A-mediated increases in serum concentrations of IL-17A and KCcompared to mice treated with PBS+IL-17A.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated soluble receptor comprising at least one ZcytoR14subunit, wherein the ZcytoR14 subunit comprises a polypeptide having anamino acid sequence of SEQ ID NO:3.
 2. An isolated soluble receptorcomprising at least one ZcytoR14 subunit, wherein the ZcytoR14 subunitcomprises amino acid residue 1 to amino acid residue 427 of SEQ IDNO:24.
 3. The soluble receptor of claim 1 or claim 2, wherein thesoluble receptor comprises two ZcytoR14 subunits, wherein said subunitsare linked together by a polypeptide linker.
 4. The soluble receptor ofclaim 3, wherein the polypeptide linker has about 100 to 240 amino acidresidues.
 5. An isolated soluble receptor comprising ZcytoR14, whereinZcytoR14 comprises a polypeptide having a sequence of amino acidresidues as shown in SEQ ID NO:3; and wherein said soluble receptorreduces the pro-inflammatory activity of either IL-17A (SEQ ID NO:14) orIL-17F (SEQ ID NO:16).
 6. The soluble receptor of claim 5, wherein saidsoluble receptor reduces the pro-inflammatory activity of both IL-17A(SEQ ID NO:14) or IL-17F (SEQ ID NO:16).
 7. An antibody or antibodyfragment that binds to a polypeptide comprising a sequence of amino acidresidues as shown in SEQ ID NO:2; and wherein said antibody or antibodyfragment reduces the pro-inflammatory activity of either either IL-17A(SEQ ID NO:14) or IL-17F (SEQ ID NO:16).
 8. The antibody or antibodyfragment according to claim 7, wherein the antibody or antibody fragmentreduces the pro-inflammatory activity of both IL-17A (SEQ ID NO:14) andIL-17F (SEQ ID NO:16).
 9. The antibody or antibody fragment according toclaim 8, wherein the or antibody fragment is (a) a polyclonal antibody,(b) a murine monoclonal antibody, (c) a humanized antibody derived from(b), (d) an antibody fragment, or (e) a human monoclonal antibody. 10.The antibody or antibody fragment according to claim 8, wherein theantibody further comprises a radionuclide, enzyme, substrate, cofactor,fluorescent marker, chemiluminescent marker, peptide tag, magneticparticle, drug, or toxin.
 11. The antibody of claim 9, wherein theantibody further comprises PEGylation.
 12. The antibody or antibodyfragment according to claim 8, wherein the or antibody fragment is (a) apolyclonal antibody, (b) a murine monoclonal antibody, (c) a humanizedantibody derived from (b), (d) an antibody fragment, or (e) a humanmonoclonal antibody.
 13. The antibody or antibody fragment according toclaim 8, wherein the antibody further comprises a radionuclide, enzyme,substrate, cofactor, fluorescent marker, chemilurninescent marker,peptide tag, magnetic particle, drug, or toxin.
 14. The antibody ofclaim 12, wherein the antibody further comprises PEGylation.
 15. Amethod of reducing IL-17A-induced or IL-17F-induced inflammationcomprising administering to a mammal with inflammation an amount of acomposition of an antibody according to claim 7 sufficient to reduceinflammation.
 16. A method of reducing IL-17A-induced or IL-17F-inducedinflammation comprising administering to a mammal with inflammation anamount of a composition of a ZcytoR14 soluble receptor according toclaim 1 sufficient to reduce inflammation.
 17. A method of treating amammal afflicted with an inflammatory disease in which IL-17A or IL-17Fplays a role, comprising: administering an antagonist of IL-17A orIL-17F to the mammal such that he inflammation is reduced, wherein theantagonist comprises (i) an antibody, antibody fragment, or bindingpolypeptide that specifically binds a polypeptide or polypeptidefragment of ZcytoR14 (SEQ ID NO:2), or (ii) a polypeptide or polypeptidefragment of ZcytoR14 (SEQ ID NO:3); and wherein the inflammatoryactivity of either IL-17A (SEQ ID NO:14) or IL-17F (SEQ ID NO:16) isreduced.
 18. The method of claim 17, wherein the disease is asthma. 19.The method of claim 17, wherein the disease is a chronic inflammatorydisease.
 20. The method of claim 19, wherein the disease is a chronicinflammatory disease comprising inflammatory bowel disease, ulcerativecolitis, Crohn's disease, arthritis, atopic dermatitis, or psoriasis.21. The method of claim 17, wherein the disease is an acute inflammatorydisease.
 22. The method of claim 21, wherein the disease is an acuteinflammatory disease comprising endotoxemia, septicernia, toxic shocksyndrome or infectious disease.
 23. The method of claim 17, wherein theantibody, antibody fragment, or binding polypeptide further comprises aradionuclide, enzyme, substrate, cofactor, fluorescent marker,chemiluminescent marker, peptide tag, magnetic particle, drug, or toxin.24. A method of treating a mammal afflicted with an inflammatory diseasein which IL-17A and IL-17F plays a role, comprising: administering anantagonist of both IL-17A and IL-17F to the mammal such that theinflammation is reduced, wherein the antagonist comprises (i) anantibody, antibody fragment, or binding polypeptide that specificallybinds a polypeptide or polypeptide fragment of ZcytoR14 (SEQ ID NO:2) or(ii) a polypeptide or polypeptide fragment of ZcytoR14 (SEQ IID NO:3);and wherein the inflammatory activity of both IL-17A (SEQ ID NO:14) andIL-17F (SEQ ID NO:16) is reduced.
 25. The method of claim 24, whereinthe disease is asthma.
 26. The method of claim 24, wherein the diseaseis a chronic inflammatory disease.
 27. The method of claim 26, whereinthe disease is a chronic inflammatory disease comprising inflammatorybowel disease, ulcerative colitis, Crohn's disease, arthritis, atopicdermatitis, or psoriasis.
 28. The method of claim 24, wherein thedisease is an acute inflammatory disease.
 29. The method of claim 28,wherein the disease is an acute inflammatory disease comprisingendotoxemia, septicemia, toxic shock syndrome or infectious disease. 30.The method of claim 24, wherein the antibody, antibody fragment, orbinding polypeptide further comprises a radionuclide, enzyme, substrate,cofactor, fluorescent marker, chemiluminescent marker, peptide tag,magnetic particle, drug, or toxin.
 31. A method of treating apathological condition in a subject associated with ZcytoR14 activitycomprising administering an effective amount of the soluble receptor ofclaim 1, thereby treating said pathological condition.
 32. The method ofclaim 31, wherein said pathological condition is asthma.
 33. The methodof claim 31, wherein said pathological condition is a chronicinflammatory condition.
 34. The method of claim 33, wherein said chronicinflammatory condition comprising inflammatory bowel disease, ulcerativecolitis, Crohn's disease, arthritis, atopic dermatitis, or psoriasis.35. The method of claim 31, wherein said pathological condition is anacute inflammatory condition.
 36. The method of claim 35, wherein saidacute inflammatory condition comprises endotoxemia, septicemia, toxicshock syndrome, or infectious disease.
 37. A method of treating a mammalafflicted with an inflammatory disease in which ZcytoR14 plays a role,comprising: administering an antagonist of ZcytoR14 to the mammal suchthat the inflammation is reduced, wherein the antagonist comprises anantibody, antibody fragment, ZcytoR14 soluble receptor or bindingpolypeptide that specifically binds a polypeptide or polypeptidefragment of ZcytoR14 (SEQ ID NO:2); and wherein the inflammatoryactivity is reduced.
 38. The method of claim 37, wherein the disease isasthma.
 39. The method of claim 37, wherein the disease is a chronicinflammatory disease.
 40. The method of claim 39, wherein the disease isa chronic inflammatory disease comprising inflammatory bowel disease,ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis, orpsoriasis.
 41. The method of claim 37, wherein the disease is an acuteinflammatory disease.
 42. The method of claim 41, wherein the disease isan acute inflammatory disease comprising endotoxemia, septicemia, toxicshock syndrome or infectious disease.
 43. The method of claim 37,wherein the antibody, antibody fragment, or binding polypeptide furthercomprises a radionuclide, enzyme, substrate, cofactor, fluorescentmarker, chemiluminescent marker, peptide tag, magnetic particle, drug,or toxin.
 44. The method of claim 37, wherein the antibody, antibodyfragment, or binding polypeptide further comprises PEGylation.